The effect of human thyroid-stimulating hormone (hTSH) on progesterone (P4) secretion during initial luteinization and subsequent prolactin (Prl)-mediated steroidogenesis by cultured rat granulosa cells was studied. Granulosa cells, obtained from pregnant mare's serum gonadotropin (PMSG)-treated immature female rats, were preincubated for 1, 3, 6, 12, or 24 h in control medium lacking added hormones or in medium containing 1.0 microgram/ml human chorionic gonadotropin (hCG) or hTSH, and maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine (bPrl). Indices of luteotropic stimulation were provided by: 1) elevated P4 concentrations determined by radioimmunoassay of spent media samples; and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation after 7 days of culture. Progesterone levels in media from cultures exposed to hCG for 24 h were twofold higher than control cultures, whereas those in media from cultures preincubated in hTSH for 24 h were fourfold higher than control levels. Cultures preincubated in 1.0 microgram/ml hCG for as little as 1 h and then maintained for 6 days in Prl secreted significantly more P4 than did control cultures also maintained with Prl for 6 days. Cultures preincubated in hTSH required a 24-h exposure before a significant increase in Prl-mediated P4 secretion was observed. Intensity of cytoplasmic osmiophilia correlated directly with P4 concentration. These results suggest that: 1) hTSH has the ability to promote P4 secretion during initial luteinization and to regulate subsequent Prl-mediated steroidogenesis by cultured rat granulosa cells; and 2) the mechanism by which hTSH stimulates Prl-mediated P4 secretion in this model system may differ from that of hCG.