The purpose of this study was to determine the effects of di(n-butyl) phthalate (DBP) administration on male reproductive organ development in F1 Sprague-Dawley rats following in utero exposure. During gestation days (GD) 10-19, pregnant rats were administered daily, orally, DBP at 250, 500, or 700 mg/kg or flutamide (1, 12.5, or 25 mg/kg/d) as a positive control. The male offspring were sacrificed at 31 d of age. DBP and flutamide dose-dependently significantly increased the incidence of hypospadias and cryptorchidism in F1 male offspring. The weights of testes and accessory sex organs (epididymides, seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles (LABC), and Cowper's glands) were significantly reduced in DBP-treated animals. Furthermore, cauda agenesis of epididymides and ventral prostate atrophy were observed in high-dose 700-mg/kg DBP males. Anogenital distance (AGD) and levels of dihydrotestosterone (DHT) and testosterone were significantly decreased in the DBP (700 mg/kg/d)-treated groups. In particular, the expression of androgen receptor (AR) and 5α-reductase type 2 in the proximal penis was markedly depressed following administration of DBP (700 mg/kg/d) or flutamide (25 mg/kg/d). The expression of sonic hedgehog (Shh) in the urethral epithelium of the proximal penis was significantly less in the DBP (700 mg/kg/d)- or flutamide (25 mg/kg/d)-treated groups. In addition, DBP dose-dependently significantly increased the expression of estrogen receptor (ER α) in the undescended testis. Data demonstrated that in utero exposure to DBP produced several abnormal responses in male reproductive organs, and these effects may be due to disruption of the stage-specific expression of genes related to androgen-dependent organs development.