Abstract The results of two-year oral toxicity studies with Ponceau 3R using two strains of rats have been presented. Dosage levels were 5.0, 2.0, 1.0, and 0.5% for Osborne-Mendel rats and one dosage level of 2.0% for Bethesda Black rats. Preliminary results of the continuing dog and mouse studies are reported. Ponceau 3R at 5.0, 2.0, 1.0, and 0.5% of the diet produced high mortality, growth inhibition, and increased weight of the liver and kidneys. Malignant liver tumors occurred in both strains of rats at all dietary levels of Ponceau 3R, statistically significant ( P < 0.01) in comparison with the controls at the 5.0% level of the Osborne-Mendel rats and the 2.0% level of the Bethesda Black animals. Incidence of benign liver tumors was also significant for all test levels. Marked weight loss and high mortality have been observed in dogs fed Ponceau 3R at dosage levels of 2.0% and 1.0%. After a feeding period of 32 weeks, changes in the liver were the most pronounced tissue changes. Increased mortality of C3HeB Fe mice has been observed on 2.0% and 1.0% feeding levels. Hepatic adenomas and carcinomas have been observed on the 2.0% level.