Steroidogenesis is a major function of the developing follicle. However, little is known about the stage of onset of steroid regulatory proteins during follicular development in sheep. In this study, several steroidogenic enzymes were studied by immunohistochemistry and/or in situ hybridization; cytochrome P450 side chain cleavage (P450_scc), cytochrome P450 17α-hydroxylase (17αOH), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 aromatase (P450_arom), steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), and LH receptor (LH-R). To define the stages of follicular growth, ovarian maps were drawn from serial sections of ovine ovaries, and follicles were located and classified at specific stages of growth based on morphological criteria. In this way, the precise onset of gene expression with respect to stages of follicular growth for all these proteins could be observed. The key findings were that ovine oocytes express StAR mRNA at all stages of follicular development and that granulosa cells in follicle types 1–3 express 3β-HSD and SF-1. Furthermore, the onset of expression in theca cells of StAR, P450_scc, 17αOH, 3β-HSD, and LH-R occurred in large type 4 follicles just before antrum formation. This finding suggests that although the theca interna forms from the type 2 stage, it does not become steroidogenically active until later in development. These studies also confirm that granulosa cells of large type 5 follicles express SF-1, StAR, P450_scc, LH-R, and P450_arom genes. These findings raise new questions regarding the roles of steroidogenic regulatory factors in early follicular development.