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dent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3– derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to ex-tend lamellipodia, as well as abnormal filopodia, exten-sions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpasta-tin-overexpressing cell lines fail to spread and assembl