Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca 2/ /calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca 2/ / calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca 2/ -dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca 2/ -and phorbol dibutyrate/1-oleoyl-2-acetyl-snglycerol-dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca 2/ , phorbol dibutyrate, or 1-oleoyl-2-acetyl-snglycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca 2/ /calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32 P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.