Male F-344 rats were fed a diet containing 2% di-(2-ethylhexyl)phthalate (DEHP) for 95 weeks. Liver nodules and/or hepatocellular carcinomas (HCC) developed in 6/10 rats fed DEHP and none were found in controls (P less than 0.005 by chi 2 test). All the nodules and HCC were negative for gamma-glutamyl transpeptidase. In the non-tumorous portions of liver, the hepatocytes contained an increased number of peroxisomes and extensive accumulation of lipofuscin. By immunocytochemical analysis, the liver peroxisomes in rats treated chronically with DEHP had visually detectable decrease in the H2O2-degrading catalase and increase in H2O2-producing fatty acyl-CoA oxidase. These results show that higher dietary level of DEHP, which causes substantially greater degree of peroxisome proliferation than the 1.2% dietary level used in the National Toxicology Program bioassay (1982, Publication no. NTP-80-37, Tech. Report Series No. 217), can induce liver tumors in male rats.