Phthalates are known to elicit marked effects on the developing male reproductive tract as evidenced by hypospadias. Recently, transforming growth factor-beta1 (TGF-beta1) was postulated to play an essential role in the development of genital tubercles (GT), and TGF- beta1 was found to act as a phthalates-responsive gene. In this study, the effects of di(2-ethylhexyl) phthalate (DEHP) were examined on the expression of TGF-beta1 in fetal mice, as GT development is dependent upon this factor. Pregnant C57BL/6 mice were exposed to corn oil or DEHP (100, 200, or 500 mg/kg/d orally) from embryonic day 12 (ED12) to ED17. Data showed a significant inhibition of male fetal GT development following DEHP treatment. Hypospadic-like urethral orifice and abnormal urethra were evaluated macroscopically and by histology at ED19. By using reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, the expression of TGF-beta1 was upregulated in DEHP-treated mice. These results suggest that hypospadias may be induced by DEHP exposure involving modification of TGF-beta1 levels.