The aim of the study was to explore the possibility of using human endometrial epithelial cells in serum-free culture as a sensitive assay for hormonal effects on the human endometrium. Glands were isolated following enzymatic digestion of the endometrial tissue and plated on a collagen matrix. The epithelial cells were grown in either medium containing serum or in supplemented serum-free medium. No morphologic difference was found between cells grown in these two media for up to 5 days, using either light or scanning electron microscopy. Secretion of prostaglandin F2 alpha (PGF2 alpha) in response to estradiol was not lower in serum-free medium than in medium containing serum for the first 2 days of culture, whereas secretion declined after prolonged incubation in the serum-free medium. This response to estradiol was clearly dose-dependent, and it was further enhanced by addition of arachidonic acid, the precursor for prostaglandin synthesis, to the medium. Co-culture of endometrial stromal cells did not influence the secretion of PGF2 alpha by epithelial cells. We conclude that the secretion of PGF2 alpha from primary cultures of human endometrial epithelial cells grown on collagen in serum-free medium can be used for a limited period as an assay of estrogenic effects on the human endometrium.