Granulosa cells were aspirated from small follicles from the ovaries of 3- to 6-month-old pigs, washed, and cultured in sealed spinner flasks. Medium 199 was supplemented with 10% fetal bovine serum, insulin, dexamethasone, T4, and antibiotics. FSH, LH, or hCG was added daily where appropriate. Cultures were sampled every other day for 10 days. Cells were examined microscopically, and cellular [125I]hCG binding and DNA content were measured. The cells aggregated and underwent morphological luteinization within 4 days in the absence or presence of FSH, LH, or hCG. Aggregation in culture was independent of clumping at the time of harvest. There was no measurable cell proliferation, based on the lack of mitotic figures and the lack of any increase in DNA. The average yield of luteinized cells from small follicle granulosa cells was 7% at 6 days. Cell concentration remained constant between days 6-10. Between days 0-2, [125I]iodo-hCG binding declined in all cultures. It continued to decline through day 10 in control cultures. In cultures containing FSH, binding increased to a maximum on day 6. The effect was specific for FSH. Increased binding was due to increased numbers of binding sites per cell. Equilibrium saturation binding data indicated that [125I]iodo-hCG binding to freshly harvested and cultured granulosa cells was specific, saturable, and of high affinity. The numbers of binding sites were 938 +/- 49 and 7602 +/- 156 sites/cell for control and FSH on day 6, respectively, compared to 498 +/- 14 sites/cell on day 0. The effect of FSH on the increase in binding sites was specific and dose related. Autoradiographic studies indicated a skewed normal frequency distribution of [125I]iodo-hCG-binding sites on cultured cells