When confluent keratinocyte cultures were wounded by cutting with a blade, the cells rapidly retracted from the wounded site, leaving an area denuded of cells. Within 3-4 h of wounding, keratinocytes began to migrate from the edges and gradually reepithelialized the entire denuded area. Mitomycin C did not prevent the reepithelialization but did dramatically inhibit [3H]thymidine incorporation into the leading edge of cells. These results indicate that cell proliferation was not required for reepithelialization. Using a rabbit antibody against urokinase-type plasminogen activator (u-PA) and an avidin-biotin-peroxidase detection method, we localized u-PA in the keratinocytes at the leading edge of the migrating cultures. Cytochalasin B dramatically inhibited the extent of migration and also altered cell morphology; nonetheless, urokinase was detected in the limited number of cells that moved into the wounded area, even in the presence of cytochalasin B. A small but consistent enhancement (36% +/- 9) of plasminogen activator activity was observed in the supernatant of wounded cultures. These data suggest that plasminogen activator may be involved in the migration of keratinocytes that occurs during wound healing.