Opening of dihydropyridine-sensitive voltage-dependent L-type Ca²⁺-channels (LTCCs) represents the final common pathway for insulin secretion in pancreaticβ -cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[³H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that α1C subunits (199 kDa, short form) contribute only a minor portion of the total α1 immunoreactivity in membranes and partially purified Ca²⁺-channel preparations. However, α1C forms a major constituent of (+)-[³H]isradipine-labeled LTCCs as 54% of solubilized (+)-[³H]isradipine-binding activity was specifically immunoprecipitated by α1C antibodies. Phosphorylation of immunopurified α1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-β-antibody directed against all known β-subunits. Isoform-specific antibodies revealed that these mainly corresponded to β1b- and β3-subunits. We found β2- and β4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that α1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. β1b- and β3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.