Insulin-stimulated phosphorylation of calmodulin in vivo was examined using a highly specific anti-calmodulin monoclonal antibody combined with high resolution two-dimensional gel electrophoresis. The two major isoforms of calmodulin immunoprecipitated from insulin-treated hepatocytes migrated on two-dimensional gel electrophoresis to the same position as nonphosphorylated calmodulin and calmodulin phosphorylated in vitro. Immunoblotting verified the identity of calmodulin. Insulin enhanced the phosphorylation of calmodulin 3.1 +/- 0.4-fold (mean +/- S.E., n = 10), with a stoichiometry in insulin-treated hepatocytes of 0.47 +/- 0.06 (mean +/- S.E., n = 3) mol of phosphate/mol of calmodulin. Two-dimensional phosphopeptide mapping of calmodulin immunoprecipitated from rat hepatocytes and calmodulin phosphorylated in vitro by the insulin receptor kinase or casein kinase II revealed several common phosphopeptides. The common phosphopeptides that appeared insulin-sensitive in intact cells comprised 61 and 40% of casein kinase II- and insulin receptor-catalyzed 32P incorporation into calmodulin in vitro, respectively. This suggests that casein kinase II and the insulin receptor kinase are, at least in part, responsible for insulin-stimulated phosphorylation of calmodulin in rat hepatocytes. These data indicate that phosphorylation of calmodulin in intact hepatocytes is significantly enhanced by insulin, supporting a critical role for calmodulin in insulin signal transduction.