Neuronal L-type Ca(2+) channels do not support synaptic transmission, but they play an essential role in synaptic activity-dependent gene expression. Ca(v)1.2 and Ca(v)1.3 are the two most widely expressed L-type Ca(2+) channels in neurons and have different biophysical and subcellular distributions. The function of the Ca(v) 1.3 L-type Ca(2+) channel and its cellular mechanisms in the central nervous system are poorly understood. In this study, using a yeast two-hybrid assay, we found that the N terminus of the rat Ca(v)1.3 alpha(1) subunit interacts with a partial N-terminal amino acid sequence of ryanodine receptor type 2 (RyR2). Reverse transcription-PCR and Western blot assays revealed high expression of both Ca(v)1.3 and RyR2 in the rat hippocampus. We also demonstrate a physical association of Ca(v)1.3 with RyR2 using co-immunoprecipitation assays. Moreover, immunocytochemistry revealed prominent co-localization between Ca(v)1.3 and RyR2 in hippocampal neurons. Depolarizing cells by an acute treatment of a high concentration of KCl (high-K, 60 mm) showed that the activation of L-type Ca(2+) channels induced RyR opening and led to RyR-dependent Ca(2+) release, even in the absence of extracellular Ca(2+). Furthermore, we found that RyR2 mRNA itself is increased by long term treatment of high-K via activation of L-type Ca(2+) channels. These acute and long term effects of high-K on RyRs were selectively blocked by small interfering RNA-mediated silencing of Ca(v)1.3. These results suggest a physical and functional interaction between Ca(v)1.3 and RyR2 and important implications of Ca(v)1.3/RyR2 clusters in translating synaptic activity into alterations in gene expression.