Granulosa cells from human preovulatory follicles were cultured under serum-free conditions to investigate the presence of immunoreactive insulin-like growth factor binding protein-3 (IGFBP-3). IGFBP-3 levels were measured by a radioimmunoassay developed against the acid-stable subunit of the protein. The antiserum had no cross-reactivity to the low molecular weight GH-independent IGFBP-1. Granulosa luteal cells exhibited a continuous release of IGFBP-3 into the culture medium during the whole time (6 days) of the incubation. A dose-dependent increase in IGFBP-3 was observed when the cells were treated by dibutyryl cAMP. Cycloheximide suppressed almost completely both the basal and the stimulated production of IGFBP-3. The smallest effective dose of dibutyryl cAMP enhancing the progesterone release was lower than that for IGFBP-3. The different time course of IGFBP-3 and progesterone secretion to dibutyryl cAMP treatment, as well as the failure of progesterone to elicit IGFBP-3 increase alone, do not support the participation of progesterone in the IGFBP-3 production of granulosa cells. It is concluded that 1. immunoreactive IGFBP-3 is produced by cultured granulosa luteal cells; 2. its synthesis is regulated by physiological intracellular mechanisms.