Cell migration requires the regulated and dynamic turnover of adhesive complexes. We have previously demonstrated that the calcium-dependent protease, calpain, regulates the organization of adhesive complexes and cell detachment during cell migration. Evidence is now provided that inhibiting calpain through over-expression of the endogenous inhibitor of calpain, calpastatin, and pharmacological inhibitors results in an inhibition of adhesive complex disassembly with stabilization of GFP-vinculin and GFP/RFP-zyxin at the cell periphery. Calpain was also required for the microtubule-mediated turnover of adhesive complex sites after nocodazole wash-out, suggesting that calpain may mediate focal complex disassembly downstream of microtubules. Using dual imaging of RFP-zyxin and GFP-alpha-actinin, we observed a temporal and spatial relationship between alpha-actinin localization to focal contacts and the subsequent disassembly or translocation of RFP-zyxin containing focal complexes in areas of cell retraction. Calpain inhibition disrupted alpha-actinin localization to zyxin-containing focal contacts and focal complex disassembly or translocation to the cell center. In addition, disrupting alpha-actinin localization to focal complexes through expression of the alpha-actinin rod domain, but not the head domain, resulted in inhibition of focal adhesion disassembly similar to calpain inhibition. Our studies suggest a novel mechanism of action whereby calpain may modulate alpha-actinin localization into focal complexes and their subsequent disassembly or translocation.