Activation of myocardial -opioid receptor-protein kinase C (PKC) pathways may improve post-ischemic contractile function through a myofilament reduction in ATP utilization. To test this we first examined the effects of PKC inhibitors on -opioid receptor-dependent cardioprotection. The -opioid receptor agonist U50,488H (U50) increased post-ischemic left ventricular developed pressure and reduced post-ischemic end diastolic pressure compared to controls. PKC inhibitors abolished the cardioprotective effects of U50. To determine if -opioid-PKC-dependent decreases in Ca²⁺-dependent actomyosin Mg²⁺ATPase could account for cardioprotection, we subjected hearts to three separate actomyosin ATPase lowering protocols. We observed moderate decreases in myofibrillar ATPase were equally rdioprotective as -opioid receptor stimulation. Immunoblot analysis and confocal microscopy revealed a -opioid-induced increase in myofilament-associated PKC-, and myofibrillar Ca²⁺-independent PKC activity was increased after -opioid stimulation. This PKC-myofilament association lead to an increase in troponin I and C-protein phosphorylation. Thus we propose PKC- activation and translocation to the myofilaments causes a decrease in actomyosin ATPase which contributes to the -opioid receptor-dependent cardioprotective mechanism.