Integrin-associated focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. In vitro, cells utilize a complex temporal and spatially regulated mechanism of focal adhesion assembly and disassembly required for cell migration. Recent studies indicate that members of both calpain and caspase protease families can promote limited proteolytic cleavage of several components of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such as tumor invasion. v-Src-induced oncogenic transformation is associated with loss of focal adhesion structures and transition to a less adherent, more motile phenotype, while inactivating temperature-sensitive vSrc in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that v-Src-induced disassembly of focal adhesions is accompanied by calpain-dependent proteolysis of focal adhesion kinase. Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion disruption, loss of substrate adhesion, and cell migration. In contrast, focal adhesion loss during detachment and apoptosis induced after switching off temperature-sensitive v-Src in serum-deprived transformed cells is accompanied by caspase-mediated proteolysis of focal adhesion kinase. Thus, calpain and caspase differentially regulate focal adhesion turnover during Src-regulated cell transformation, motility, and apoptosis.The transforming viral Src gene (v-src) is associated with classic characteristics of oncogenic cell transformation, including deregulated growth control, cell rounding, and substrate detachment resulting from adhesion loss and disruption of the actin cytoskeleton (1-6). The stability of the actin cytoskeleton and adhesive properties of cells are mediated, at least in part, by focal adhesion complexes (7,8). Dynamic regulation of these adhesive links through assembly of focal adhesions at the leading edge of cells, coordinated with focal adhesion disassembly at the trailing edge, plays a key role in controlling cell migration (9). The mechanisms regulating turnover of focal adhesions are not well understood. However, a recent study suggests that proteolysis of specific components of the focal adhesion complex by the calpain family of proteolytic enzymes promotes disassembly of smooth muscle focal adhesions in response to collagen fragments (10). Furthermore, calpain activity has been implicated in promoting migration of Chinese hamster ovary cells (11). The calpains are defined as a well conserved family of intracellular, nonlysosomal calciumdependent cysteine proteases consisting of two ubiquitously expressed calpain isozymes, -calpain (calpain-I) and m-calpain (calpain-II) and several tissue-specific isoforms (12)(13)(14). Colocalization of calpain II with focal adhesion structures (15) and the identification of several focal adhesion protei...