To examine the possibility that insulin might stimulate calmodulin phosphorylation in intact cells, we compared autoradiographs of two-dimensional gels of [35S]methionine- and 32P-labeled proteins from 3T3-L1 adipocytes, before and after immunoprecipitation with anti-calmodulin antiserum. Insulin stimulated the phosphorylation of one or two proteins of approximately 22 kDa and pI 4.6; this increased phosphorylation was accompanied by an apparent shift in the position of the analogous [35S]methionine-labeled proteins towards the anode. In contrast, insulin had no effect on the phosphorylation state of another protein of 18-22 kDa and pI 4.6. This protein was very heavily labeled with [35S]methionine, co-migrated exactly with purified calmodulin, reacted specifically with two anti-calmodulin antibodies by Western blotting, and was specifically immunoprecipitated with the anti-calmodulin antiserum. Similar amounts of [35S]methionine-labeled calmodulin were immunoprecipitated from control and insulin-stimulated cells, arguing against the possibility that insulin-stimulated phosphorylation of calmodulin changed its affinity for the antibody. We conclude that calmodulin is phosphorylated to a negligible extent in serum-deprived 3T3-L1 adipocytes and that insulin does not stimulate its phosphorylation under conditions in which it stimulates the phosphorylation of one or more neighboring proteins.