Prolonged in utero exposure of fetal male rats to di-butyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes, and therefore reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semi-quantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including StAR, SR-BI, CYP11A1, and CYP17A1A1, that are down-regulated following an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by electrophoretic mobility shift assay, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein beta (c/ebp ) and steroidogenic factor-1 (SF-1). ChIP confirmed declines in DNA-protein interactions of c/ebp in DBP treated animals, while SF-1 was observed to be reduced in both Di-ethyl phthalate (DEP; non-toxic) and DBP (toxic) treatments. These results suggest inhibition of c/ebp , and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. Additionally, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure.