Calponin is a basic, approximately 34,000 M(r), smooth muscle-specific protein which is developmentally expressed in up to four isoforms. Calponin binds very strongly to actin in a Ca(2+)-independent manner and is localized to the thin filaments in smooth muscle, where it is present at a stoichiometry of 1 mol calponin/7 mol actin. The interaction of calponin with actin inhibits the actomyosin MgATPase (cross-bridge cycling rate) without affecting myosin phosphorylation. The calponin-actin interaction is blocked and calponin-mediated inhibition of the actomyosin MgATPase is reversed upon phosphorylation of calponin by either PKC or CaM kinase II; these properties are restored upon dephosphorylation of calponin by a type 2A protein phosphatase. Consistent with these in vitro findings, calponin is phosphorylated in intact smooth muscle in response to contractile stimuli. The increasing body of evidence, both in vitro and in vivo, strongly supports calponin phosphorylation-dephosphorylation as a thin filament-linked regulatory system in smooth muscle.