Regulation of calcium current through L-type calcium channels (ICa,L) of the guinea pig taenia coli smooth muscle cell (SMC) membrane by cyclic nucleotides and protein kinase C (PKC) was studied using a voltage-clamp technique with intracellular dialysis or membrane patch perforation with amphotericin B. Non-selective blockers of serine/threonine kinase, staurosporine and H-7 reduced the ICa,L amplitude in a dose-dependent manner. Dose-dependent suppression of ICa,L was also produced by a selective PKC blocker, chelerythrine, and a cAMP-and cGMP-dependent protein kinase (PKA, PKG), blocker H-8. Forskolin, which increases the intracellular level of cAMP, as well as membrane-permeant cAMP analogs, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, exerted complex effects on ICa,L. The latter increased at their concentrations below 10 μM and decreased at their higher concentrations. 8-Bromo-cGMP reduced ICa,L in all cases. Addition of 50 μM GTPγS to the micropipette solution caused a marked and slowly developing increase in ICa,L. 8-Bromo-cAMP (1 μM) increased ICa,L by 30%, both in the control and during the action of GTPγS. The blockade of PKC by 10 μM chelerythrine removed the effect of GTPγS on ICa,L. The results suggest that basal activity of L-type calcium channels in SMC of the guinea pig taenia coli depends on PKC- and PKA-dependent phosphorylation. PKC can increase the ICa,L amplitude provided G proteins are activated. cAMP at low concentrations likewise increases ICa,L (probably through activation of PKA). PKG apparently mediates ICa,L drops evoked by cAMP at high concentrations and by cGMP.