In previous studies we have shown that voltage dependent L-type Ca²⁺ channels (Cav) in portal vein myocytes are enhanced when muscarinic M2 receptors are activated with acetylcholine. Current stimulation was coupled to the G-protein subunit G along with the downstream mediators phosphoinositide 3 kinase (PI3K), PKC and c-Src. The present study was designed to determine whether the same second messenger pathway could be identified when exogenous recombinant G subunits are introduced into cells. Smooth muscle myocytes were freshly isolated from rabbit portal vein and Cav currents recorded using the patch clamp technique. Dialysis of cells with recombinant G (50 nM) significantly increased Cav currents (141%). Nifedipine (1 µM) reduced both control and stimulated currents by approximately 90%. The enhancement of current by G was equivalent to that produced by acetylcholine (142%) whereas the PKC activator phorbol 12,13-dibutyrate (PdBu) gave rise to greater current stimulation (192%). Current stimulation with G, acetylcholine and PdBu were not associated with changes in the voltage dependence of activation or inactivation. The PI3K inhibitor, LY 294002 (20µM) reduced peak currents by 32% in cells dialyzed with G whereas the inactive analogue LY 303511 resulted in a small but significant reduction in current (12%). The c-Src inhibitor, PP2 (1µM) also significantly reduced currents (34%) whereas the inactive analogue PP3 was without effect. These data provide further evidence for the hypothesis that G leads to stimulation of Cav currents in rabbit portal vein myocytes via a signaling pathway that includes PI3K, PKC and c-Src.