Single-chain urokinase-type plasminogen activator (sc-uPA) is one of the key serine proteases involved in modulating cellular and extracellular matrix responses during tissue remodeling. Sc-uPA is composed of three domains: aminoterminal fragment (ATF), kringle domain, and carboxyterminal fragment (CTF). sc-uPA is readily cleaved into these three domain fragments in vitro, each of which is biologically active; however, their roles in the microenvironment of the vessel wall are poorly understood.The purpose of this study was to determine the role of each domain of sc-uPA on vascular smooth muscle cell (SMC) proliferation and migration.SMCs were cultured in vitro. Assays of DNA synthesis, cell proliferation, and migration were performed in response to sc-uPA, ATF, kringle, and CTF in the presence and absence of the plasmin inhibitors epsilon-aminocaproic acid (EACA) and aprotinin, the Galphai inhibitor pertussis toxin, and the mitogen-activated protein kinase 1 (the upstream regulator of the extracellular-signal regulated kinase [ERK]) inhibitor PD98059.sc-uPA produced dose-dependent increases in DNA synthesis and cell proliferation. These responses were dependent on the CTF domain and were sensitive to plasmin inhibitors, pertussis toxin, and PD98059. Sc-uPA also induced SMC migration, which could be elicited by both ATF and kringle. Migration to sc-uPA, ATF, and kringle was both pertussis toxin and PD98059 sensitive, but importantly was plasmin-independent.sc-uPA induces SMC proliferation and migration, which are domain-dependent and mediated in part by Galphai-linked, ERK-dependent processes, while only the mitogenic response is protease dependent. These findings suggest that migration is linked to a G-protein coupled nonprotease receptor, while proliferation is associated with a G-protein coupled protease receptor.