Two members of the GATA-binding family of transcription factors, GATA-4 and GATA-6, are expressed in the vertebrate ovary. To gain insight into the role of these factors in ovarian cell differentiation and function, we used in situ hybridization to determine the patterns of expression of GATA-4 and GATA-6 in mouse ovary during development and in response to hormonal stimulation. GATA-4 messenger RNA (mRNA) was first evident in the ovary around the time of birth. In the adult ovary, abundant GATA-4 mRNA was detected in granulosa cells of primary and antral follicles, with lesser amounts of GATA-4 message detected in theca cells, germinal epithelium, and interstitial cells. Little or no GATA-4 mRNA was found in corpus luteum. GATA-6 message exhibited a different distribution in the ovary, with abundant expression evident in both granulosa cells and corpora lutea. Stimulation of 3-week-old females with PMSG or estrogen enhanced follicular expression of GATA-4 and GATA-6 transcripts. Subsequent induction of ovulation with human CG resulted in a decrease in GATA-4 mRNA expression in granulosa cells, whereas GATA-6 mRNA expression persisted in granulosa cells after ovulation and in corpora lutea. Moreover, follicular apoptosis was associated with a decrease in the expression of GATA-4 but not GATA-6 message. Stimulation of cultured gonadal cell lines with FSH resulted in increased expression of GATA-4 message, whereas GATA-6 mRNA expression was not affected. In light of these findings, the established role of other GATA-binding proteins in hematopoetic cell differentiation and apoptosis, and the presence of conserved GATA motifs in the promoters of genes expressed selectively in ovary, we propose that GATA-4 and GATA-6 play distinct roles in follicular development and luteinization.