In utero exposure to di(n-butyl) phthalate (DBP) leads to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists. DBP demonstrates no affinity for the androgen receptor, but rather leads to diminished testosterone production by the fetal testis. The purpose of this study was to determine the onset and reversibility of DBP effects on the fetal testis and to determine at a functional level the points in the cholesterol transport and steroidogenesis pathways affected by DBP. Starting at gestational day (gd) 12, pregnant rats were gavaged daily with 500 mg/kg DBP or corn oil control. Significant decreases in testosterone production and mRNA expression of scavenger receptor B1, P450_SCC, steroidogenic acute regulatory protein, and cytochrome p450c17 were observed as early as gd 17. Testosterone, mRNA, and protein levels remained low 24 h after withdrawal of DBP treatment but increased 48 h after cessation of DBP exposure. In another experiment, pregnant dams were treated with DBP until gd 19, with the start of DBP treatment moved 1 d later into gestation for each treatment group, with the final group dosed only on gd 19. Significant decreases in testosterone, mRNA expression, and protein expression were evident as early as 3 h after treatment with DBP, with full repression apparent 24 h after treatment. Using a testis explant system, we determined that DBP treatment led to diminished transport of cholesterol across the mitochondrial membrane as well as diminished function at each point in the testosterone biosynthesis pathway except 17β-hydroxysteroid dehydrogenase. The transcriptional repression caused by DBP does not appear to be mediated via interference with steroidogenic factor-1 as determined by reporter assays. We conclude that high-dose DBP exposure leads to rapid and reversible diminution of the expression of several proteins required for cholesterol transport and steroidogenesis in the fetal testis, resulting in decreased testosterone synthesis and consequent male reproductive maldevelopment.