The present study demonstrates that the α, ε, and ι isozymes of protein kinase C (PKC) are translocated to particulate fractions from the cytosol during brief intervals of global ischemia as well as reperfusion of ischemic rat myocardium. In contrast, phorbol ester treatment of perfused hearts resulted in the translocation of the α, δ, and ε isozymes of PKC to particulate fractions. Additionally, the α, δ, and ε isozymes of PKC are translocated to particulate fractions in phorbol ester-stimulated, isolated adult rat cardiac myocytes. Concomitant with the translocation of PKC isozymes to particulate fractions during myocardial ischemia, increased protein phosphorylation was observed, which was blocked by pretreatment of hearts with the selective PKC inhibitor bisindolylmaleimide I (50 nM). In particular, ischemia resulted in the phosphorylation of 26-, 20-, and 17-kDa particulate-associated proteins. Taken together, the present findings are the first to demonstrate that specific PKC isozymes are translocated to particulate fractions in the ischemic and the reperfused ischemic rat heart, resulting in the phosphorylation of specific particulate-associated proteins.