Two affinity-purified polyclonal antibodies have been generated that differentially recognize two mol wt (Mr) variants of prostaglandin H synthase (PGS) in the rat ovary: antibody-2 recognized PGS of 72,000 Mr (PGS72), and antibody-3 recognized PGS of 69,000 Mr (PGS69). Immunoblot analyses showed that PGS72 was rapidly induced by LH in granulosa cells of preovulatory (PO) follicles and was associated with the increased production of prostaglandins (PGs) obligatory for ovulation. PGS72 was low (negligible) in other ovarian tissues, including PO follicles, corpora lutea, and interstitium. In contrast, PGS69 was constitutively present in small antral and PO follicles (primarily in thecal cells), was unaffected by LH, and was found at higher levels in corpora lutea throughout pregnancy and in the ovarian interstitium. PGS69 (but not PGS72) was also detected by immunoblots in rat adrenal glands, heart, uterus, and kidney. Immunofluorescent localization of PGS72 and PGS69 to ovarian tissue sections confirmed the cell-specific distribution of PGS observed by immunoblot analyses of cell extracts. Immunofluorescent detection of PGS72 required methanol fixation, whereas PGS69 was also observed with paraformaldehyde fixation and Triton X-100 permeabilization, further suggesting biochemical differences in these molecules. Immunoreactive PGS69 in PO follicles, thecal cells, and granulosa cells was associated with low amounts of indomethacin-sensitive production of PGs by these tissues in vitro, which was unaffected by inhibitors of transcription or translation. In contrast, stimulation of PGs in PO follicles by LH in vitro correlated with the marked induction of PGS72, but not PGS69, and was sensitive to both transcriptional and translational inhibitors. Collectively, these studies provide the first evidence that the rat ovary contains two immunologically distinct forms and Mr variants of PGS, each of which is selectively regulated by hormones, localized to specific cell types, differentially sensitive to inhibitors of transcription/translation, and differentially solubilized for immunocytochemical localization.