Individual mouse strains may differ significantly in terms of behavior and cognitive function. Hippocampal gene expression profiling on several mouse strains has been carried out and points toward substantial strain-specific variation of more than 200 genes including components of major signaling pathways involved in neuronal information storage. Strain-specific hippocampal protein expression, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry has been chosen to address this question by determining strain-dependent expression of signaling proteins in hippocampi of four inbred and one outbred mouse strain. Forty-six spots corresponding to 37 different signaling proteins have been analyzed and quantified. Statistical analysis revealed strain-dependent expression of serine/threonine protein phosphatase 1, serine/threonine protein phosphatase 2A, large GTP binding protein OPA1, guanine nucleotide-binding protein beta, putative GTP-binding protein Ran, receptor of activated protein kinase C1, WASP-family protein member 1, voltage-dependent anion channel 2 and 14-3-3 protein gamma. Differential expression of signaling proteins in the hippocampus may contribute to the molecular understanding of strain-dependent behavioral and cognitive performance. Moreover, these data highlight the importance of the genetic background for the analysis of signaling pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.