One of the initial stages of adipogenesis is migration of preadipo- cytes of mesenchymal origin into cell clusters to form primitive fat organs. The serine protease inhibitor plasminogen activator inhibi- tor-1 (PAI-1) is synthesized and released from human adipose tissue ex vivo and regulates smooth muscle and endothelial cell migration in vitro, but its role in adipose tissue is not known. We investigated the role of PAI-1 in cultures of human preadipocytes from men and women of various ages and body mass indexes. Human preadipocytes ex- pressed the messenger ribonucleic acid for PAI-1 and released sig- nificant quantities of PAI-1 protein into the medium. As PAI-1 reg- ulates motility through the interaction of vitronectin with its receptor, the integrin aVb3, we identified this receptor in human preadipocytes. Flow cytometric analysis indicated that human preadipocytes express the vitronectin receptor aVb3 in a similar pattern as human umbilical vein endothelial cells. Functional studies indicated that active, but not latent, PAI-1 inhibited preadipocyte attachment to vitronectin with an IC50 of 13.3 nmol/L, and preincubation of vitronectin-coated Transwells with active PAI-1 prevented preadipocyte migration. Vi- tronectin was identified in homogenates of the stromal-vascular frac- tion of human adipose tissue, but was absent from human adipocytes and cultured preadipocytes. These data indicate that human pre- adipocyte migration is regulated through the endogenous expression of PAI-1 and aVb3 integrin, a novel autocrine mechanism for poten- tially regulating cell cluster formation in adipogenesis. (J Clin Endocrinol Metab 85: 2609 -2614, 2000)