The protein kinase C activator, (-)-indolactam, has been shown to enhance reactivity of arterioles by a mechanism not requiring an increase in intracellular Ca++ (Ca++i). The aim of this study was to characterize the Ca++ requirement for indolactam-induced contraction of resistance vessels. Studies were performed in small mesenteric arteries (diameter, 260 +/- microm), using intact segments or after permeabilization with alpha-toxin (500 U/ml). Additional studies were preformed using isolated smooth muscle cells to allow electrophysiological assessment of the effect of indolactam on voltage-gated Ca++ entry. Intact and permeabilized vessel segments showed dose-dependent constriction to indolactam. Studies of Fura 2-loaded vessels and permeabilized segments maintained at low Ca++i, showed that the constriction occurred without an overt increase in Ca++i. That Ca++ was required was evident by near maximal relaxation after the removal of Ca++. Patch clamp studies indicated that indolactam potentiated voltage-gated Ca++ entry; however, nifedipine (0.5 microM) and La (0.2 or 1 mM) were relatively ineffective in reversing the contraction, indicating that voltage-gated Ca++ entry was not an absolute requirement. In intact vessel segments, the myosin light chain (MLC) kinase inhibitors, ML-7 and ML-9, reversed the indolactam contraction, suggesting the requirement of the MLC chain phosphorylation pathway. Furthermore, indolactam caused by a significant increase in MLC phosphorylation in permeabilized vessels, despite clamping of Ca++i at pCa 7.0. The data are consistent with the suggestion that the protein kinase C activator, indolactam, acts to modulate the Ca++ sensitivity of the smooth muscle contractile process such that higher than expected levels of MLC phosphorylation exist for a given level of Ca++i.