Gossypol acetic acid is a polyphenolic compound present in the seed of cotton plants. Its antifertility activity by inhibition of spermatogenesis was proven in a large group of animals, including man. In the present study, the direct effect of gossypol acetic acid on collagenase isolated rat I-cells (interstitial cells) was investigated. It was shown that gossypol acetic acid depressed significantly the metabolic rate of the cells. Glucose utilization was abolished by a starting dose of 100 μg/ml. Oxygen consumption of I-cells was reduced even at a smaller dose of gossypol (50 μg/ml). At these doses, the vitality of the cells remained (proven by trypan blue exclusion test). slightly 3 β-Hydroxysteroid dehydrogenase (3 β-HSD) histochemical stain was decreased. Increasing doses of gossypol caused a marked decrease in the vitality of I-cells and a dramatic drop in histochemical stain for 3 β-HSD. The pH of the medium was not changed at any dose of treatment. In cultures of I-cells not stimulated by hCG, gossypol did not affect the tonic slow release of testosterone. Thus, gossypol acetic acid has a direct inhibitory effect on isolated rat I-cells, depressing cell metabolism. The failure of some of the other groups to show such an effect, especially in vivo, can be attributed to differences in the dose of treatment and strain of animals.