Testes from adult (90-120-day-old) rats, which had been made cryptorchid 28 days previously, were dispersed by successive treatment with trypsin, collagenase and hyaluronidase. The resulting crude cell suspension was fractionated on discontinuous Percoll density gradients to yield five distinct cell bands (1-5), at the interface between successive layers of Percoll. Crude cells and purified fractions were cultured for up to 7 days, and inhibin was subsequently measured in the media by radioimmunoassay and in vitro bioassay. Sertoli cells from density gradient bands 2 (1.03-1.04 g/ml) and 3 (1.04-1.05 g/ml) showed minimal germ cell or peritubular cell contamination, as determined by morphological and histochemical techniques. Cells from these bands secreted significantly higher levels of immunoactive inhibin/microgram DNA/48 h under both basal and either follicle-stimulating hormone (FSH)- (100 ng/ml) or dibutyryl cAMP-stimulated (100 micrograms/ml) conditions than did cells from the other bands. While there was a decline in basal secretion of inhibin with increasing duration of culture, the capacity of the purified Sertoli cells (bands 2 and 3) to respond to both FSH and dibutyryl cAMP increased over the culture period. The addition of dibutyryl cAMP (31.25-500 micrograms/ml) to the purified Sertoli cells also caused a stimulation of bioactive inhibin. Immunoactive inhibin production by purified Sertoli cells was unaffected by the addition of either rat LH (8 ng/ml) or testosterone (10(-6) M). The data describe a method for the isolation of adult Sertoli cells from cryptorchid testes, and demonstrate their responsiveness to both FSH and dibutyryl cAMP in vitro using the measurement of immunoactive inhibin as a marker of Sertoli cell function.