The physiologic function of the serpin heparin cofactor II (HCII) is not fully understood. We have hypothesized that HCII functions as an extravascular inhibitor of thrombin. Thrombin formed at a site of injury has been hypothesized to contribute to migration and proliferation of fibroblasts and smooth muscle cells involved in wound healing. To begin to test our hypothesis, we examined the immunohistochemical localization of HCII in human skin and compared it to that of the closely related serpin, antithrombin (ATIII). In skin specimens with acute wounds, there was diffuse HCII and ATIII staining in areas of hemorrhage. In healing skin wounds ATIII was primarily associated with mast cells, while HCII was associated with mononuclear phagocytes in the dermis. Blood monocytes isolated from healthy donors also stained for HCII protein. However, in situ hydridization and RT-PCR studies failed to show significant HCII mRNA expression either in macrophages in wounded skin or in peripheral blood leukocytes. HCII localization is not due to nonspecific uptake of plasma proteins, since ATIII had a very different distribution in wounded skin. These findings support the notion that HCII could function as an extravascular thrombin inhibitor and might play a role in the regulation of wound healing.