Follicular fluid obtained from large (6-12 mm) porcine follicles (LFF) was investigated to determine its stimulatory activity on progesterone secretion and on follicle stimulating hormone (FSH) induction of 125I-human chorionic gonadotropin (hCG)-luteinizing hormone (LH) binding sites in porcine granulosa cells in a 4-day culture. Incubation of granulosa cells harvested from small porcine follicles (1-2 mm) with 50% LFF led to stimulation of LH/hCG binding sites and progesterone secretion. After partial purification of pooled LFF or proteins precipitated with 90% ethanol on Sephadex G-100 the greatest stimulatory activity was found in the second protein peak eluted from the column. Chromatography of part of the active fraction on DEAE Sephacel using a continuous gradient of NH4HCO3 yielded seven protein fractions. The second fraction, which eluted early, contained the majority of the stimulatory activity which was purified about 32-fold compared to native LFF. In contrast, addition of follicular fluid recovered from small porcine follicles inhibited FSH induction of LH/hCG binding sites and progesterone secretion. It can be concluded, that maturation of granulosa cells from small follicles may be enhanced by protein(s) present in LFF, but not in fluid recovered from less mature follicles.