The cytoplasmic domains of integrin beta subunits are involved in bidirectional transmembrane signaling. We report that the cytoplasmic domain of the integrin beta 3 subunit undergoes limited proteolysis by calpain, an intracellular calcium-dependent protease. Calpain cleavage occurs during platelet aggregation induced by agonists such as thrombin. Five cleavage sites have been identified. Four of these sites (C-terminal to Thr741, Tyr747, Phe754, and Tyr759) are utilized in intact platelets and flank two NXXY motifs (Asn744-Pro-Leu-Tyr747 and Asn756-Ile-Thr-Tyr759). The fifth site (Ala735) is accessible to calpain after EDTA treatment of the alpha IIb beta 3 heterodimer. The NXXY motif is critical to the bidirectional signaling functions of beta 3 integrins and their association with the cytoskeleton. Thus, calpain cleavage of the beta 3 cytoplasmic domain may provide a means to regulate integrin signaling functions.