The mode and sites of antifertility action of gossypol were explored in Long-Evans rats for the purpose of clarifying effects on endocrine function. Mature male rats were treated every five days with gossypol acetic acid (10 mg/kg of body weight, given subcutaneously). Although no conspicuous loss in sperm motility and breeding performance occurred with this dosing regimen, even after prolonged treatment, a statistically significant reduction in serum testosterone (T) and decreases in accessory organ weights were observed after only 3 weeks. Testosterone propionate (500 micrograms daily, subcutaneously) administered concurrently with gossypol to gonadectomized rats prevented the decrease in accessory organ weight, indicating that gossypol probably affects these organs indirectly by altering circulating androgen levels. In another experiment, serum T was measured in gossypol-treated (10 mg/kg/day for 100 days, subcutaneously) and control rats before and after a single challenge dose of 20 IU of hCG. Even though prechallenge serum T levels were suppressed in gossypol-treated rats, postchallenge levels were similar to controls, indicating the absence of significant impairment in interstitial cell response to gonadotropin stimulation. There was also no evidence that the anterior pituitary gland was directly affected by gossypol treatment.