The expression of urokinase-type plasminogen activator (u-PA) and its type-1 inhibitor (PAI-1) was examined in vivo in mouse wounds by in situ hybridization and immunohistochemistry. u-PA mRNA was present in both basal and suprabasal keratinocytes in the regenerative epithelial outgrowths at the edge of the wounds. In the same area, PAI-1 mRNA was only present in the basal keratinocytes. u-PA protein was detected in keratinocytes in several layers of the epithelial outgrowth, whereas PAI-1 protein was confined to the basal keratinocytes and to the area of the basal membrane. The two proteins and their mRNA were not detected in normal epidermis or in normal-looking epidermis adjacent to the wounds. Fibroblast-like cells and fairly large stellate cells (possibly macrophages) in the granulation tissue underneath the wound contained both the two proteins and their mRNA. The large stellate cells, showing a strong hybridization signal for PAI-1 mRNA, were especially abundant at the border between the necrotic wound and the newly formed granulation tissue. The specificity of these results was supported by the use of two different non-overlapping antisense probes, sense mRNA probes, antibody preparations preabsorbed with purified proteins, and Northern analysis of tissue extracts. The localized and regulated expression of u-PA and PAI-1 seen in this study may reflect that plasminogen activation plays a role in the migration of keratinocytes and connective tissue cells during reepithelialization and tissue remodeling in wound healing.