Hippocalcin (Hpca) is a member of the neuronal calcium sensor protein family and is highly expressed in hippocampal neurons. Hpca-deficient (Hpca–/–) mice display a defect in cAMP response element-binding protein (CREB) activation associated with impaired spatial and associative memory. Here we examine the involvement of Hpca in the extracellular signal-regulated kinase (ERK) cascade leading to CREB activation, because application of PD98059, a broad ERK cascade inhibitor, has resulted in similar levels of CREB activation in Hpca–/– hippocampus. N-methyl-D-aspartate (NMDA)- and KCl-induced phosphorylation of ERK was significantly attenuated in Hpca–/– hippocampal slices, as was ionomycin-induced phosphorylation of ERK, whereas forskolin and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation yielded indistinguishable levels of ERK phosphorylation in both wild-type and Hpca–/– slices. In an in vitro reconstitution assay system, recombinant Hpca affected neither Raf-1 protein kinase activity with recombinant MEK-1 as a substrate nor MEK-1 kinase activity with ERK2 as a substrate. Activation of Ras by NMDA and KCl stimulation of hippocampal slices showed no obvious changes between the two genotypes; however, phosphorylation of Raf-1 was significantly lower in Hpca–/– slices. These results suggest that Hpca plays an important role in the activation of Raf conducted by Ras. © 2007 Wiley-Liss, Inc.