Osteopontin (OPN) is an important chemokinetic agent for several cell types. Our earlier studies have shown that its expression is essential for uridine triphosphate (UTP)-mediated migration of vascular smooth muscle cells. We demonstrated previously that the activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat OPN promoter is involved in the induction of OPN expression. In this work, using a luciferase promoter deletion assay, we identified a new region of the rat OPN promoter (-1837 to -1757) that is responsive to UTP. This region contains an NFkappaB site located at -1800 and an Ebox located at -1768. Supershift electrophoretic mobility shift assay and chromatin immunoprecipitation assays identified NFkappaB and USF-1/USF-2 as the DNA binding proteins induced by UTP, respectively, for these two sites. Using dominant negative mutants of IkappaB kinase and USF transcription factors, we confirmed that NFkappaB and USF-1/USF-2 are involved in the UTP-mediated expression of OPN. Using a pharmacological approach, we demonstrated that USF proteins are regulated by the extracellular signal-regulated kinase (ERK)1/2 pathway, just as the earlier discovered AP-1 complex, whereas NFkappaB is up-regulated through PKCdelta signals. Finally, our work suggests that the UTP-stimulated OPN expression involves a coordinate regulation of PKCdelta-NFkappaB, ERK1/2-USF, and ERK1/2/NAD(P)H oxidase AP-1 signaling pathways.