Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of cellular growth and proliferation by G1 phase arrest or apoptosis. We investigated the association of TGF-beta1 with the anti-proliferative effect of upstream stimulatory factor (USF) in Fischer rat thyroid cell line (FRTL-5) cells. [methyl-(3)H] thymidine uptake was measured after treatment of FRTL-5 cells with TGF-beta1 to identify its anti-proliferative effect. USF-1 and USF-2 proteins were in vitro translated, and an electrophoretic mobility shift assay was performed to identify the interaction between USF and the TGF-beta1 promoter. FRTL-5 cells were transfected with USF cDNA, and then the expression of TGF-beta1 was examined with Northern and Western blotting. The cell cycle-regulating proteins associated with TGF-beta1 were also measured. TGF-beta1 significantly inhibited [methyl-(3)H] thymidine uptake in FRTL-5 cells. Two specific binding sites for USF were found in the TGF-beta1 promoter: -1,846 approximately -1,841 (CACATG) and -621 approximately -616 (CATGTG). Overexpression of USF increased both the mRNA levels and protein levels of TGF-beta1. However, the expression of cyclin D1, CDK4, cyclin E, and CDK2, and the phosphorylation of retinoblastoma protein remained unchanged. Overexpression of USF in FRTL-5 cells increased the expression of TGF-beta10 through specific binding to TGF-beta1 promoter. However, the USF-induced expression of TGF-beta1 did not cause G1 arrest.