Human granulosa cells were collected from preovulatory follicles during follicular puncture for in-vitro fertilization. They were cultured in serum-free medium supplemented with ascorbic acid. Using a combination of high-performance liquid chromatography and radioimmunoassay, the oxytocin material present in the cell extracts and secreted into the medium was identified. When cells were deprived of ascorbate, intermediary forms resulting of the post-translational processing of pro-oxytocin/neurophysin were detected. These data demonstrate that oxytocin biosynthesis occurs in human granulosa cells.