Amitrole, a widely used herbicide found to produce thyroid and liver tumors in rodents and classified as possibly carcinogenic to humans, was investigated to acquire further information about its mechanism of action. A 20-hr exposure to amitrole concentrations ranging from 5.6 to 18 mM did not induce DNA fragmentation, as measured by the alkaline elution technique, in primary cultures of human thyroid follicular cells and of human liver cells. Under the same experimental conditions a minimal frequency of DNA breaks was detected in primary cultures of rat hepatocytes, but this event was presumably the unspecific consequence of a cytotoxic effect. In rats given amitrole with drinking water for 12 successive days at a daily dose of approximately 200 mg/kg, plasma levels of triiodothyronine and thyroxine displayed a progressive reduction, and a concurrent increase of both the mitotic index and frequency of S-phase cells revealing a clear-cut follicular cell hyperplasia was observed. In a group of these rats euthanized after 8 days of treatment any evidence of DNA fragmentation was absent in both thyroid and liver cells. Taken as a whole these results provide further evidence that the mechanism of amitrole carcinogenic activity is most likely nongenotoxic but due to hormone imbalance.