Effects of phosphorylation of bovine cardiac troponin T (TnT) by protein kinase C on the Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin complex and the binding of TnT to tropomyosin(Tm)-F-actin were investigated. The Ca(2+)-stimulated MgATPase of actomyosin containing phosphorylated TnT (1.8 mol of P/mol), compared with that containing unphosphorylated TnT, was decreased by up to 48%. Phosphorylation of TnT also decreased (up to 48%) its maximum binding to Tm-F-actin, which was accompanied by a decrease (up to 3.5-fold) in its apparent binding affinity. The findings indicate that the effects of phosphorylated TnT in decreasing actomyosin MgATPase might be secondary to its decreased interactions with the other components of the thin filament, representing a new mechanism underlying the negative inotropic responses of various cardiac preparations to protein kinase C-activating phorbol esters.