The modulation of transferrin secretion by FSH and epidermal growth factor (EGF) was studied in highly pure, primary cultures of immature rat Sertoli cells grown on a reconstituted basement membrane (Matrigel) in bicameral chambers. Sertoli cell purity was assessed by (1) morphometry, (2) alkaline phosphatase cytochemistry (a specific marker enzyme for peritubular cells) and (3) immunocytochemistry for the alpha-isoform of smooth muscle actin in contaminating peritubular cells. Results revealed a less than 0.5% peritubular cell contamination. During initial periods of culture with EGF or FSH alone or in combination, both EGF and FSH alone maintained transferrin secretion over basal values and their effects were additive. At subsequent times, EGF alone maintained transferrin secretion, but to less extent than did FSH alone, and inhibited significantly the ability of FSH to maintain transferrin secretion. The ratio of polarized transferrin secretion in response to FSH, EGF, or in combination was also examined. FSH significantly reversed the polarity of transferrin secretion, whereas EGF, although significantly reducing the ratio of apical to basal transferrin secretion, did not lead to a preferential basal secretion of transferrin. The change in the apical:basal transferrin secretion ratio, however, was not due to a reversal of the apically secreted transferrin towards a basal direction, but rather to an increase in the total basally secreted transferrin. The effects of cell density effects on transferrin secretion were then examined. At low cell density, the relative ability of EGF and FSH together to maintain transferrin secretion was greater than at high cell density, but overall transferrin secretion was greater as cell density increased. The inhibition of FSH by EGF on transferrin secretion was also density dependent: EGF significantly inhibited FSH effects at low cell density, but failed to do so at high cell density. These results suggest that regulation of transferrin secretion by Sertoli cells appears to be under dual control, involving both FSH and EGF and may provide an explanation for the mechanism by which EGF exerts a regulatory role in spermatogenesis.