Cultured hippocampal neurons and organotypic slices have been useful for investigating long-lasting plasticity beyond the time limit of acutely prepared slices. However, difficulties with culturing adult neurons have restricted such studies to preparations from embryonic, perinatal, and juvenile tissue. Immature hippocampal cultures not only differ in the anatomical organization and maturity of their neurons from adult tissue, but also in the mechanisms for the induction and expression of long-term potentiation (LTP). This study provides evidence that mature hippocampal cultures can retain electrophysiological properties required for long-term plasticity for several weeks in vitro. Introducing improved methods for culturing and maintaining hippocampal-entorhinal cortex slices from young adult rats (P25-30) resulted in cultures for use in long-term electrophysiological investigations. The electrophysiological properties and, in particular, the induction of LTP in mature organotypic slices were highly sensitive to dissection and tissue culture techniques. Using the modified preparation and culture protocols, cultured mature slices maintained an intact and functional trisynaptic cascade, synaptic function comparable to acute slices, as well as reliable long-term recording stability for at least 14 days in vitro. As in the adult hippocampus in vivo, LTP at the Schaffer-collateral-CA1 synapse could be induced by extracellular stimulation. Its induction was N-methyl-D-aspartate (NMDA) receptor dependent and its maintenance long-lasting (> 4 h). The development of mature slice cultures and protocols for LTP induction makes further studies investigating the mechanisms involved in the long-lasting maintenance of LTP feasible. For example, phosphorylation of the transcription factor cAMP-response element binding protein (CREB) has been implicated in synaptic plasticity and long-term memory, and its sustained activation has been proposed to be required for the maintenance of late-LTP (L-LTP). In the present work, the level of CREB phosphorylation was determined for individual neurons in mature organotypic hippocampal slices after LTP was induced by stimulating the CA1 area. Confocal imaging was used to determine the ratio between nonphosphorylated and phosphorylated CREB (pCREB) revealing the extent of CREB phosphorylation at a single-cell resolution. The activation of CREB after LTP induction was compared to cAMP-activation after bath application of forskolin. An increase in cAMP by forskolin resulted in a persistent and uniform increase of the pCREB/CREB immunofluorescence ratio in the entire hippocampal principal neuron population. High-frequency tetanization (100Hz) in the CA1 area resulted in long-lasting LTP accompanied by a significant increase in the pCREB/CREB ratio, which continued to increase in parallel with the increased duration of LTP. Specific for CA1 cells following tetanization was a marked variability of CREB phosphorylation between adjacent cells throughout the duration of LTP. Only LTP-inducing stimuli translated synaptic input into varied degrees of CREB phosphorylation, and resulted in the continued increase of the proportion of nuclear CREB phosphorylation in parallel to the maintenance of long-lasting LTP irrespective of the initial level of activation. Activity-dependent CREB activation was specific for CA1 neurons, whereas CA3 and dentate neurons remained at baseline levels indicating that antidromic stimulation was not sufficient for inducing CREB phosphorylation. In addition, 100 Hz stimulation in the presence of an NMDA receptor antagonist resulted in a short-lasting posttetanic potentiation and an unchanged pCREB/CREB ratio revealing that both CREB phosphorylation and LTP induction in mature slices required NMDA receptor activation. This study supports the hypothesis that CREB may play a role in the late phases of LTP and provides evidence that molecular and electrophysiological plasticity can be studied in parallel in mature cultured tissue, which can be maintained in culture without a loss in hippocampal cell function or stability.