The ovulatory process in mammals involves a substantial increase in the metabolism of steroids and eicosanoids in response to a surge in LH or to an injection of hCG into experimental animals. This study provides evidence that the ovulatory stimulus causes induction of the gene for 3α-hydroxysteroid dehydrogenase (3α-HSD), an enzyme that belongs to several oxidoreductase superfamilies that affect steroid and eicosanoid metabolism. Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction (PCR) differential display to detect gene expression in the stimulated ovarian tissue. One of the PCR primer sets differentially amplified a cDNA fragment that is 52.3% homologous with a 3α-HSD gene in rat liver. Northern analyses revealed that maximum transcription was at 8 h after the animals had been treated with hCG. The Northerns also indicated that the 3α-HSD cDNA probe cross-hybridized with as many as six different bands of mRNA on the blots. In situ hybridization localized 3α-HSD mRNA in the granulosa and thecal layers of mature follicles and in newly formed corpora lutea at 24 h after the ovulatory stimulus. In conclusion, gene(s) for 3α-HSD are transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin. A possible function of the oxidoreductase enzyme that is translated from the 3α-HSD mRNA may be to reduce the toxic aldehyde and ketone components of the steroids and eicosanoids that accumulate in the mammalian ovary at the time of ovulation.