The effect of protein kinase C (PKC) activation on cardiac mechanoenergetics is not fully understood. To address this issue, we determined the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on isolated rat hearts. Hearts were exposed to PMA with or without pretreatment with the PKC inhibitor chelerythrine. Contractile efficiency was assessed as the reciprocal of the slope of the linear myocardial O₂ consumption (O₂) pressure-volume area (PVA) relation. PMA decreased contractility (E_max; 30 ± 8%; P < 0.05) and increased coronary perfusion pressure (+58 ± 11%; P < 0.01) without altering left ventricular end-diastolic pressure. Concomitantly, PMA decreased PVA-independent O₂ [nonmechanical energy expenditure for excitation-contraction (E-C) coupling and basal metabolism] by 28 ± 8% (P < 0.05) and markedly increased contractile efficiency (+41 ± 8%; P < 0.05) in a manner independent of the coronary vascular resistance. Basal metabolism was not affected by PMA. Chelerythrine abolished the PMA-induced vasoconstriction, negative inotropy, decreased PVA-independent O₂, and increased contractile efficiency. We conclude that PKC-mediated phosphorylation of regulatory proteins reduces O₂ via effects on both the contractile machinery and the E-C coupling.