Pancreatic islet cytosol contains a calcium-calmodulin dependent protein kinase that can mediate the phosphorylation of an endogenous protein that has an Mr of 57 000, as well as exogenous muscle pyruvate kinase (subunit Mr, 57000). EGTA and trifluoperazine decreased the phosphorylation. Alkaline inactivation of pyruvate kinase made it a better substrate for the kinase. As in rat islet cytosol, rabbit islet cytosol catalyzed the phosphorylation of a 57 000 Mr protein in the presence of calcium and calmodulin. This phosphoprotein was immunoprecipitated with anti-pyruvate kinase antibody. This is consistent with the idea that the 57 000 Mr phosphoprotein in islet cytosol is the subunit of pyruvate kinase. The paper following this paper shows that the kinetic and immunologic properties of the islet pyruvae kinase indicate it is the M₂ isoenzyme and that its phosphorylation does not affect its catalytic activity.