This study investigated regulation of L- type calcium channels ( Cav 1.2b) by acetylcholine ( ACh) in rabbit portal vein myocytes. Whole- cell currents were recorded using 5 mmol/ L barium as charge carrier. ACh ( 10 mumol/ L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4- DAMP ( 100 nmol/ L) but was blocked by the M2 receptor antagonist methoctramine ( 5 mumol/ L). The classical and novel protein kinase C ( PKC) antagonist calphostin C ( 50 nmol/ L) abolished ACh responses, whereas the classical PKC antagonist Go6976 ( 200 nmol/ L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3- kinase ( PI3K) inhibitor LY294002 ( 20 mumol/ L), by the c- Src inhibitor PP2 ( 10 mumol/ L) ( but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G- protein subunit Gbetagamma. Cells dialyzed with c- Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c- Src is downstream of PI3K. Phorbol 12,13- dibutyrate ( PDBu, 0.1 mumol/ L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Go6976. The PDBu response was also reduced by PP2, and the PP2- insensitive component was blocked by Go6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c- Src. PDBu stimulates the novel PKC/ c- Src pathway along with a second pathway that is independent of c- Src and involves a classical PKC.