Focal adhesion kinase (125 kDa form; pp125FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signal transduction. We have identified a novel means of pp 125FAK regulation in human platelets, in which this kinase undergoes sequential proteolytic modification from the native 125 kDa form to 90, 45 and 40 kDa fragments in thrombin-, collagen- and ionophore A23187-stimulated platelets. The proteolysis of pp125FAK was prevented by pretreating platelets with the calpain inhibitors calpeptin or calpain inhibitor-1, and was reproduced in vitro by incubating immunoprecipitated pp125FAK with purified calpain. Proteolysis of pp125FAK resulted in a dramatic reduction in its autokinase activity and led to its dissociation from the cytoskeletal fraction of platelets. These studies define a novel signal-terminating role for calpain, wherein proteolytic modification of pp125FAK attenuates its autokinase activity and induces its subcellular relocation within the cell.