Estradiol biosynthesis is a key biochemical trait of developing follicles. To study its regulation in equine follicles, the objectives of this study were to clone and determine the structure of equine cytochrome P450 aromatase (P450AROM), and characterize the regulation of P450AROM and P450 17␣-hydroxylase/C17-20 lyase (P45017␣) messenger RNAs (mRNAs) in vivo in equine preovulatory follicles isolated during hCGinduced ovulation. Two distinct P450AROM complementary DNAs (cDNAs) were isolated from an equine preovulatory follicle cDNA library. One clone was 2682 bp in length and included 115 bp of 5Ј-untranslated region (UTR), 1509 bp of open reading frame encoding a well conserved 503-amino acid protein, and 1058 bp of 3Ј-UTR. Its 5Ј-most region represented the equine homolog of exon 1f, previously designated brain specific. The other cDNA clone encoded a truncated protein and contained a distinct 5Ј-UTR characteristic of transcripts derived from promoter II, previously identified as the predominant ovarian mRNA. Northern blot analyses were performed using preovulatory follicles obtained during estrus between 0 -39 h after the administration of hCG and with corpora lutea isolated on day 8 of the estrous cycle (day 0 ϭ day of ovulation). The results showed a biphasic regulation of P450AROM mRNA expression: levels were highest in follicles at 0 h post-hCG, decreased significantly during the ovulatory process at 12 and 24 h (P Ͻ 0.05), and increased again between 30 -39 h post-hCG and in corpora lutea. When oligonucleotides specific for P450AROM mRNA variants were used as probes, a novel switching phenomenon was observed. Promoter II-derived transcripts accounted for the message present in follicles at 0 h post-hCG and in corpora lutea, whereas promoter 1f-derived mRNA was expressed exclusively during the ovulatory process (30 -39 h post-hCG). Levels of P45017␣ mRNA were high in follicles at 0 h, but significantly decreased after hCG treatment (P Ͻ 0.05), with lowest levels in follicles at 36 and 39 h post-hCG and in corpora lutea. Northern blots performed on isolated cellular preparations revealed that P450AROM and P45017␣ transcripts were localized exclusively in granulosa cells and theca interna, respectively. Equine aromatase promoters II and 1f were cloned from a genomic library, and putative transcription start sites were characterized by primer extension assays. Sequence analyses identified distinct potential regulatory elements in each promoter. Thus, this study identifies a novel aromatase promoter-switching phenomenon in equine granulosa cells during follicular luteinization and provides a new model in which aromatase promoter switching is induced in vivo. (Endocrinology 140: 4133-4141, 1999)