This report describes the first study to correlate the ultrastructure of long-term monolayer cultures of human luteal cells with their secretion of relaxin and progesterone under basal and human chorionic gonadotropin-stimulated conditions. In culture from 14 to 28 days, cells from both corpora lutea of the menstrual cycle and corpora lutea of pregnancy took on characteristics of granulosa luteal cells, particularly after exposure to human chorionic gonadotropin. Relaxin was detectable in the luteal cell cultures only at early time points. The effect of human chorionic gonadotropin on media relaxin levels differed in cells of the cycle and cells of pregnancy. In the cells of the cycle, relaxin was detectable only on day 2 and was decreased by human chorionic gonadotropin (p < 0.01). In cells of pregnancy, relaxin was detectable for the first 4 days of culture and was not affected by human chorionic gonadotropin. Progesterone was detectable in all the luteal cell cultures and was enhanced by human chorionic gonadotropin (10 and 50 IU/ml) after 24 days of exposure. At earlier time points in both the luteal cells of the cycle and the luteal cells of pregnancy, the human chorionic gonadotropin-induced increases in progesterone levels were not as consistent. However, in all cases of progesterone enhancement, smooth-surfaced endoplasmic reticulum was increased in the human chorionic gonadotropin-treated cells compared with corresponding controls, consistent with more active steroid production. In addition, gap junctions, considered to be responsive to trophic hormones, were increased in the treated cells. In conclusion, this long-term monolayer culture of human luteal cells, as monitored by ultrastructural and hormonal changes, retained the differentiated function of progesterone secretion and exhibited responsiveness to human chorionic gonadotropin. Therefore, morphologic and functional aspects of progesterone secretion may be investigated more closely with use of this long-term luteal cell culture system.