Granulosa cells (GC) harvested from large (6-12 mm) follicles of cycling pigs obtained at the abattoir were incubated in the absence or presence of insulin and/or human chorionic gonadotropin (hCG). The total accumulation of progesterone and cAMP in the tissue plus medium was quantified by radioimmunoassay of hexane extracts and trichloroacetic acid extracts, respectively, and was related to cellular DNA content. Insulin (1 microgram/ml) had little effect on progesterone accumulation by freshly harvested GC incubated for up to 26 h at 37 degrees C. After preincubation of GC for 16 h in the absence of hormones, insulin (650 ng/ml) only enhanced subsequent basal progesterone accumulation after an additional 22 h. However, preincubation of cells for 20 h in the presence of 1 microgram/ml of insulin increased subsequent progesterone accumulation in both the absence and the presence of hCG (50 ng/ml). The effect of a 20-h preincubation with insulin on subsequent progesterone accumulation was related to the insulin concentration. At a maximally effective concentration of 5 micrograms/ml during the preincubation period, insulin increased subsequent basal and hCG-stimulated progesterone accumulations more than twofold. The concentrations of insulin in the preincubation medium that produced half maximal stimulation (ED50) were 300 and 200 ng/ml for subsequent basal and hCG-stimulated progesterone accumulations, respectively. After correction for loss of insulin immunoreactivity in the incubation medium, the ED50 levels were 60 and 50 ng/ml, respectively. Insulin elevated hCG-stimulated progesterone accumulation over the entire range of hCG concentrations; the effect on hCG-stimulated cAMP production was evident only at higher hCG concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)