The effect of agonists on palmitoylated proteins was examined in platelets prelabeled with [3H]palmitic acid. Non-reduced gels revealed major labeled proteins with masses from 30-38 kDa. One of these proteins was modified by A23187, which led to a loss of radioactivity, and PMA, which altered its electrophoretic mobility. A possible link between the A23187-induced loss of label associated with the protein and the activation of calpain was suggested by the following experiments. (1) There was a good correlation between the loss of label and the proteolysis of proteins in A23187-activated platelets. (2) The permeant calpain inhibitor, E64d, blocked the loss of label as well as the proteolysis of proteins. (3) The loss of label also occurred in a Triton lysate, where calpain was known to be activated. The effect of PMA on the palmitoylated protein was observed only in prelabeled platelets. The protein kinase inhibitor, staurosporine, abolished the PMA-induced platelet aggregation as well as the mobility shift of the labeled protein.