Smooth muscle cell proliferation and migration into neointima are hallmarks of atherogenesis. However, mechanisms responsible have not yet been fully elucidated. One potential mediator of both smooth muscle cell proliferation and migration is activation of plasminogen by activators bound to receptors on cells within the vessel wall. To determine whether vascular smooth muscle cells within atheroma express the receptor for urokinase-type plasminogen activator (uPA-R), we characterized atheroma in cholesterol-fed New Zealand White rabbits and human subjects by immunostaining. Intense immunostaining of uPA-R was observed throughout the neointima in both rabbit and human atherosclerotic lesions with the use of a monoclonal antibody to uPA-R. uPA-R was not detectable in normal arterial tissues. uPA-R was localized to macrophages and neointimal smooth muscle cells identified by immunostaining in serial sections. Furthermore, uPA-R protein in extracts from atheroma was present in at least a ninefold greater quantity compared with extracts from normal vessels, as shown by Western blotting. Expression of uPA-R mRNA in migrating vascular smooth muscle cells did not increase significantly. Thus, altered posttranscriptional regulation may be contributing to the increased uPA-R. In vitro, antibodies to uPA-R delayed the migration of cultured vascular smooth muscle cells. Our results suggest that increased cell-surface uPA-R contributes to pericellular proteolysis and consequently increased neointimalization secondary to increased vascular smooth muscle cell migration in atheroma.