The di-tert-butyl-substituted hydroxyanisoles 2,5-di-tert-butyl-4-hydroxyanisole (2,5-DTBHA) and 3,5-di-tert-butyl-4-hydroxyanisole (3,5-DTBHA) are contaminants of the commercial antioxidant butylated hydroxyanisole. The tumorigenicity of 2,5-DTBHA, 3,5-DTBHA, 2,5-di-tert-butyl hydroquinone (2,5-DTBHQ), a demethylated analog of 2,5-DTBHA, and 3-tert-butyl-4-hydroxyanisole (3-BHA) were investigated. Dietary 3-BHA, which has been shown previously to induce neoplastic lesions in the hamster forestomach, was found to cause the development of forestomach papilloma in 75% of the hamsters in 24 weeks at 1% addition to the diet. General thickening of the forestomach lining with moderate to severe hyperkeratosis and hyperplasia were observed in 14 (88%) of the 16 hamsters in this group. Inflammation was generally found in the forestomach of these animals. Similar observations were found in 14 (88%) of the 16 hamsters in the group that was fed 1% DTBHQ. Severe hyperplasia and papilloma found in the forestomach of these hamsters were similar in severity to those in the 3-BHA group. Unlike the 3-BHA and 2,5-DTBHQ groups, the forestomachs of 1% 2,5-DTBHA and 3,5-DTBHA treated animals were not different from the hamsters in the control group. Only mild hyperplasia at or near the limiting ridge was found in the forestomach of some of these animals. Hyperkeratosis or inflammation were not observed. Steric factors may play a role in the lack of tumorigenicity of 2,5-DTBHA and 3,5-DTBHA, while the formation of a stable free radical from 2,5-DTBHQ may be the activated species responsible for its tumorigenicity.