We have previously identified and purified a splenocyte-derived factor (PSF) that stimulates the accumulation of progesterone and 20α-dihydroprogesterone (20α-OH-P) in rat ovarian granulosa cells independently of FSH. In the present study, time course experiments comparing the response to PSF with that to FSH revealed that PSF-stimulated progesterone accumulation was slower than that of FSH, but PSF-stimulated 20α-OH-P accumulation had a time course similar to that of FSH. To determine the basis for the slower progesterone response to PSF, the effect of these two agents on each step of the steroidogenic pathway was assessed. First, to examine whether PSF-stimulated cholesterol mobilization was limiting, cultured granulosa cells were treated with 22(R)-hydroxycholesterol. While both FSH-and PSF-stimulated progesterone and 20α-OH-P accumulation approximately doubled, the overall time courses did not change indicating that cholesterol availability was not the factor limiting the response to PSF. Next, PSF and FSH induction of steroidogenic enzyme activities and messenger RNAs were compared. While FSH-stimulated cytochrome P450 side chain cleavage enzyme (SCC) activity rapidly increased (peaking at 2 days) and then slowly declined, PSF-stimulated SCC activity gradually increased over 5 days to approximately 35% of the maximal activity stimulated by FSH. PSF also induced slower increases in P450scc mRNA levels than did FSH. In addition, PSF stimulated 3β-hydroxysteroid dehydrogenase (3β-HSD) activity more slowly than did FSH, but after 3 days of culture, PSF-stimulated activity was significantly higher than that induced by FSH. A similar pattern was also observed with 3β-HSD mRNA levels. In contrast, FSH stimulated only minimal increases in 20α-hydroxysteroid dehydrogenase (20α-HSD), whereas PSF stimulated larger and more rapid increases in 20α-HSD activity, which were maximal by 3 days. These studies indicate that the slower onset of PSF-stimulated progesterone production is due, at least in part, to slower induction of mRNAs for P450scc and 3β-HSD leading to slower increases in the activities of their respective enzymes. Additionally, the higher 20α-OH-P levels produced by PSF may be due to greater induction of 20α-HSD activity. © 1994.