Experiments were designed to determine the effects of feeding the methylxanthines caffeine, theobromine, or theophylline to 4- to 6-week-old males rats at a dietary level of 0.5 percent for periods ranging from 14 to 75 weeks. In the first two experiments, Osborne-Mendel rats were fed the test substances alone or in combination with sodium nitrite to test the hypothesis that these amines might nitrosate in vivo to produce toxic nitrosamine compounds. The compounds failed to produce neoplastic or preneoplastic lesions, but a significant positive finding was the occurrence of severe bilateral testicular atrophy with aspermatogenesis or oligospermatogenesis in 85-100 percent of the rats fed caffeine or theobromine. In a third experiment the methylxanthines were fed to Holtzman rats for 19 weeks to determine whether testicular atrophy would be induced in a second strain of rat. The testicular effects were similar to those in Experiments I and II but were more pronounced. Caffeine and theobromine induced testicular injury in nearly all rats. Theophylline induced severe testicular atrophy in 14 percent of the rats, mild to moderate atrophy in 71 percent, and had no effect in 15 percent. The relative testicular toxicity of the methylxanthines was caffeine, most potent; theobromine, slightly less potent; and theophylline, considerably less potent. Somewhat variable atrophic changes of the accessory sexual organs (epididymis, prostate, and seminal vesicles) accompanied the testicular changes. Cytogenetic analysis of testes from caffeine- or theophylline-treated rats revealed a significantly reduced number of mitotic cells in the caffeine-treated group. Plasma testosterone concentrations were significantly elevated in the theobromine group and somewhat elevated in the caffeine-treated group; this correlated morphologically with an apparent hyperplasia of interstitial cells in severely atrophied testes in these groups. Plasma cholesterol concentrations were significantly increased in the caffeine and theobromine groups. Possible sites and mechanisms of action of the methylxanthines in the induction of testicular atrophy and impaired spermatogenesis are discussed.