The broad spectrum protease urokinase-type plasminogen activator (uPA) has been implicated in muscle regeneration in vivo as well as in myogenic proliferation and differentiation in vitro. These processes are known to be modulated by basic fibroblast growth factor (FGF-2) and serum. We therefore investigated the mechanism(s) underlying the regulation of uPA expression by these two stimuli in proliferating and differentiating myoblasts. The expression of uPA mRNA and the activity of the uPA gene product were induced by FGF-2 and serum in proliferating myoblasts. uPA induction occurred at the level of transcription and required the uPA-PEA3/AP1 enhancer element, since deletion of this site in the full promoter abrogated induction by FGF-2 and serum. Using L6E9 skeletal myoblasts, devoid of endogenous FGF receptors, which have been engineered to express either FGF receptor-1 (FGFR1) or FGF receptor-4 (FGFR4), we have demonstrated that both receptors, known to be expressed in skeletal muscle cell precursors, were able to mediate uPA induction by FGF-2, whereas serum stimulation was FGF receptor-independent. The induction of uPA by FGF-2 and serum in FGFR1- and in FGFR4-expressing myoblasts required the mitogen-activated protein kinase pathway, since treatment of cells with a specific inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase-2 kinase, PD98059, blocked uPA promoter induction. Although FGF-2 and serum induced uPA in proliferating myoblasts, their actions on cell-cell contact-induced differentiating myoblasts differed dramatically. FGF-2, but not serum, repressed uPA expression in differentiation-committed myoblasts, and these effects were also shown to occur at the level of uPA transcription. Altogether, these results indicate a dual regulation of the uPA gene by FGF-2 and serum, which ensures uPA expression throughout the whole myogenic process in different myoblastic lineages. The effects of FGF-2 and serum on uPA expression may contribute to the proteolytic activity required during myoblast migration and fusion, as well as in muscle regeneration.