L-type voltage dependent Ca(2+) channels (L-VDCCs; Ca(v)1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G(q) enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G(q)-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α(1C), whereas known smooth muscle short-NT isoforms were inhibited by PKC and G(q) activators. We report a novel regulation of the long-NT α(1C) isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G(q)--but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α(1C)-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α(1C) sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G(q)-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α(1C) are involved.