Heterologous expression studies have shown that the activity of voltage-gated Ca2+ channels is regulated by their beta subunits in a beta subunit isoform-specific manner. In this study we therefore investigated if one or several beta subunit isoforms associate with L-type Ca2+ channels in different regions of mammalian brain. All four beta subunit isoforms (beta1b, beta2, beta3, and beta4) are expressed in cerebral cortex as shown in immunoblots. Immunoprecipitation of (+)-[3H]isradipine-labeled L-type channels revealed that the majority of beta subunit-associated L-type channels was associated with beta3 (42 +/- 8%) and beta4 (42 +/- 7%) subunits, whereas beta1b and beta2 were present in a smaller fraction of channel complexes. beta3 and beta4 were also the major L-type channel beta subunits in hippocampus. In cerebellum beta1b, beta2, and beta3 but not beta4 subunits were expressed at lower levels than in cortex. Accordingly, beta4 was the most prominent beta subunit in cerebellar L-type channels. This beta subunit composition was very similar to the one determined for 125I-omega-conotoxin-GVIA-labeled N-type and 125I-omega-conotoxin-MVIIC-labeled P/Q-type channel complexes in cerebral cortex and cerebellum. Our data show that all four beta subunit isoforms associate with L-type Ca2+ channels in mammalian brain. This beta subunit heterogeneity may play an important role for the fine tuning of L-type channel function and modulation in neurons.