Di(n-butyl) phthalate (DBP) alters male reproductive development by decreasing testicular testosterone (T) production when fetuses are exposed on gestation days (GD) 12-21. Previous studies have shown altered gene expression for enzymes in the T biosynthetic pathway following exposure to DBP. The objectives of this study were to develop a more detailed understanding of the effect of DBP on steroidogenesis, using a robust study design with increased numbers of dams and fetuses, compared with previous studies, and to explore messenger RNA (mRNA) expression for other critical genes involved in androgen biosynthesis and signaling. Additionally, immunohistochemical localization of protein expression for several key genes was performed to further confirm mRNA changes. Fetal Leydig cell lipid levels were also examined histochemically, using oil red O. Six to seven pregnant Crl:CD(SD)BR rats per group were gavaged with corn oil or DBP at 500 mg/kg/day on GD 12-19. Testicular RNA isolated from three randomly selected GD 19 fetuses per litter was used for real-time RT-PCR for the following genes: scavenger receptor class B-1 (SRB1), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17, 17beta-hydroxysteroid dehydrogenase (17beta-HSD), androgen receptor (AR), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), stem cell factor tyrosine kinase receptor (c-kit), stem cell factor (SCF), proliferating cell nuclear antigen (PCNA), and testosterone-repressed prostate message-2 (TRPM-2). mRNA expression was downregulated for SRB1, StAR, P450scc, 3beta-HSD, P450c17, and c-kit following DBP exposure, and TRPM-2 was upregulated. 17beta-HSD, AR, LHR, FSHR, and PCNA were not significantly changed. Immunohistochemical staining for c-kit was seen in fetal Leydig cells, which has not been previously reported. Downregulation of most of the genes in the T biosynthetic pathway confirms and extends previous findings. Diminished Leydig cell lipid content and alteration of cholesterol transport genes also support altered cholesterol metabolism and transport as a potential mechanism for decreased T synthesis following exposure to DBP.