The preovulatory LH rise is the physiological trigger of follicular luteinization, a process during which the synthesis of progesterone is markedly increased. To study the control of follicular progesterone biosynthesis in mares, the objectives of this study were to clone and characterize the equine cholesterol side-chain cleavage cytochrome P450 (P450_scc) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-isomerase (3β-HSD), and describe the regulation and cellular localization of their transcripts in equine follicles during hCG-induced ovulation. Complementary DNA cloning and primer extension analyses revealed that the equine P450_scc transcript is composed of a 5′-untranslated region (UTR) of 52 nucleotides, an open reading frame (ORF) of 1560 nucleotides, and a 3′-UTR of 225 nucleotides, whereas the equine 3β-HSD mRNA consists of a 5′-UTR of 61 nucleotides, an ORF of 1119 nucleotides, and a 3′-UTR of 432 nucleotides. The equine P450_scc and 3β-HSD ORF encode 520 and 373 amino acid proteins, respectively, that are highly conserved (68–79% identity) when compared to homologs of other mammalian species. Northern blot analyses were performed with preovulatory follicles isolated 0, 12, 24, 30, 33, 36, and 39 h post-hCG, and corpora lutea obtained on day 8 of the cycle. Results showed that levels of P450_scc mRNA in follicular wall (theca interna with attached granulosa cells) decreased after hCG treatment (30–39 h versus 0 h post-hCG, P < 0.05), and increased again after ovulation to reach their highest levels in corpora lutea (P < 0.05). Northern blots on isolated cellular preparations revealed that theca interna was the predominant site of P450_scc expression in follicles prior to hCG (P < 0.05). However, transcript levels decreased in theca interna between 30–39 h (P < 0.05) and increased in granulosa cells at 39 h (P < 0.05), making the granulosa cell layer the predominant site of P450_scc expression at the end of the ovulatory process. A different pattern of regulation was observed for 3β-HSD, as transcript levels remained constant throughout the luteinization process (P > 0.05). Also, in contrast to other species, expression of 3β-HSD mRNA in equine preovulatory follicles was localized only in granulosa cells and not in theca interna. Thus, this study characterizes for the first time the complete structure of equine P450_scc and 3β-HSD mRNA and identifies novel patterns of expression and regulation of these transcripts in equine follicles prior to ovulation.