Summary—Epithelial glands were isolated from guinea‐pig endometrium. In order to reduce the requirement for a serum supplement and the contamination by non epithelial cells in primary culture, various coatings of the culture dishes were tested using serumfree Ham's F12 containing defined chemicals including 17β‐estradiol. while epithelial glands seeded on culture dishes coated with Matrigel, a basement membrane matrix‐failed to spread, they formed on poly‐d‐lysine plus serum‐coated dishes, a subconfluent monolayer (5–7 days) enriched in cytokeratin‐immunostained cells (78%). Cells from subconfluent primary cultures, obtained on poly‐d‐lysine plus serum‐coated dishes in serum‐free hormonally defined medium, were passaged on Matrigel‐coated dishes in serum‐free hormonally defined medium. These subcultures contained, at confluence (4–5 days), a high percentage (>95%) of cytokeratin‐immunostained cells. These monolayers consisted of well‐differentiated cells which exhibited ultrastructural features characteristics of endometrial epithelial cells. Moreover, these confluent cells contained 50% immunostained nuclei for progesterone receptors. Progesterone receptor amounts decreased in confluent subcultures treated with progesterone and became undectable after longterm treatment, suggesting responsiveness of these cells to progesterone. This culture system provides a well‐defined model for the study of protein synthesis and secretion by endometrial