The aim of this study was to determine 1) the time of onset and cellular localization of gene expression for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase/Δ⁵,Δ⁴ isomerase (3β-HSD), and the cytochrome P450 enzymes for cholesterol side-chain cleavage (P450_scc), 17α-hydroxylase (P450_17αOH), and aromatase (P450_arom) during gonadal development; and 2) the amount of progesterone, androstenedione, testosterone, and 17β-estradiol present in the fetal sheep gonad. Fetuses were collected on Days 24, 26, 28, 30, 32, 35, 40, 55, and 75 of gestation, and gene expression was determined by in situ hybridization. The steroid content of gonads collected on Days 30, 35, 55, and 75 of gestation was determined by RIA. Developing gonads collected from both male and female fetuses were steroidogenically active around the time of morphological sexual differentiation. In the female, the steroidogenic cells were initially located at the boundary of the cortex and medulla but become increasingly restricted to the mesonephric-derived cell streams. In the male, once tubules were identifiable, steroidogenesis was restricted to the interstitial regions. Interestingly, expression of both SF-1 and 3β-HSD was observed prior to morphological sexual differentiation. In addition, expression of both of these genes was more widespread than the other genes in both males and females.