Learning and long-term memory are thought to involve temporally defined changes in gene expression that lead to the strengthening of synaptic connections in selected brain regions. We used cDNA microarrays to study hippocampal gene expression in animals trained in a spatial discrimination-learning paradigm. Our analysis identified 19 genes that showed statistically significant changes in expression when comparing Nai;ve versus Trained animals. We confirmed the changes in expression for the genes encoding the nuclear protein prothymosin(alpha) and the delta-1 opioid receptor (DOR1) by Northern blotting or in situ hybridization. In additional studies, laser-capture microdissection (LCM) allowed us to obtain enriched neuronal populations from the dentate gyrus, CA1, and CA3 subregions of the hippocampus from Nai;ve, Pseudotrained, and spatially Trained animals. Real-time PCR examined the spatial learning specificity of hippocampal modulation of the genes encoding protein kinase B (PKB, also known as Akt), protein kinase C(delta) (PKC(delta)), cell adhesion kinase(beta) (CAK(beta), also known as Pyk2), and receptor protein tyrosine phosphatase(zeta/beta) (RPTP(zeta/beta)). These studies showed subregion specificity of spatial learning-induced changes in gene expression within the hippocampus, a feature that was particular to each gene studied. We suggest that statistically valid gene expression profiles generated with cDNA microarrays may provide important insights as to the cellular and molecular events subserving learning and memory processes in the brain.