Recently, we have demonstrated that myocardial sarcolemma is predominantly comprised of plasmalogen molecular species and that the plasmalogen metabolite 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) accumulates during myocardial ischemia despite substantial decreases in 1,2-diacyl-sn-glycerol (DAG) content. To elucidate the physiological significance of AAG accumulation during myocardial ischemia, rabbit myocardial protein kinase C was partially purified by DE-52 and high-performance hydroxylapatite chromatographies, and the potency of AAG as an activator of myocardial protein kinase C was assessed. Both AAG and 1-O-alkyl-2-acyl-sn-glycerol are potent activators of myocardial protein kinase C with obligatory requirements for physiological increments in free Ca2+ concentration. In contrast, a substantial amount of myocardial protein kinase C activity elicited by DAG was calcium independent. Concentration dependence of ATP for protein kinase C-mediated phosphorylation was identical utilizing either ether-linked diglycerides or DAG as activators, with maximal phosphorylation manifest at ATP concentrations two orders of magnitude less than those found in ischemic myocardium. Thus accumulation of AAG in ischemic myocardium in conjunction with increases in intracellular free Ca2+ concentration may synergistically activate protein kinase C and therefore modulate phosphorylation of proteins in specific subcellular loci.