To be able to study the control mechanisms for human luteal function, a system was designed to maintain human luteal cells in culture. Collagenase dispersed cells of human corpora lutea of the menstrual cycle and pregnancy were maintained as nonolayer cultures for 26 days. Progesterone (P) and relaxin (R) in culture media were measured by radioimmunoassay. Both menstrual cycle and pregnancy luteal cells secreted P for 26 days. hCG increased P secretion by menstrual cycle luteal cells, but not by pregnancy luteal cells. R was not detected in media of menstrual cycle luteal cell controls, nor in media of cells incubated with hCG. R was detected in media of pregnancy luteal cells for 6 days. Addition of hCG caused a significant increase in media R levels only on day 2 of culture. These studies show that human luteal cells can be maintained as viable monolayer cultures for at least 26 days and these cultures can be used to study control of human luteal function.