The effects of the chronic administration of methyl-deficient, amino acid-defined diets on liver tumor formation were examined in male weanling C3H/HeN mice previously treated with a single ip injection of 0 or 150 mg diethylnitrosamine/kg body weight [(DENA) CAS: 55-18-5]. Five diets were used: diet 1, adequate; diet 2, devoid of both methionine and choline; diet 3, devoid of methionine only; diet 4, devoid of choline only; and diet 5, devoid of methionine, choline, folic acid, and vitamin B12. Equimolar homocystine replaced methionine in all methionine-devoid diets. All diets were administered for 1 year. No hepatocellular carcinomas and only 3 liver adenomas were seen among the 129 animals at risk in the 5 groups that had received no DENA. Among the DENA-treated groups fed diets 1-4, the incidence of hepatocellular carcinomas in the mice at risk averaged 40%, with no significant differences noted among groups. A relatively low incidence of liver carcinomas (10%) was seen among DENA-treated mice subsequently fed diet 5; it could be ascribed to the enhanced mortality seen in these animals due to the dietary deficiencies. Lung tumors were seen in 44% of the DENA-treated mice surviving more than 35 experimental weeks and in only 2.5% of the corresponding DENA-untreated animals. Feeding diet 2, deficient in methionine and choline, to male C3H mice for 5-20 weeks decreased the hepatic ratio of S-adenosylmethionine (CAS: 29908-3-0) to S-adenosylhomocysteine (979-92-0) relative to that observed in mice fed the adequate diet 1. The 5-methyldeoxycytidine [(5-MC) CAS: 838-07-3] contents of liver DNA in animals fed diet 2 for 5, 10, and 20 weeks, however, were not significantly different from the corresponding levels in diet 1-fed mice. The results indicate that a methionine- and choline-deficient dietary regimen that lowers the 5-MC levels in DNA and enhances liver tumor formation in male F344 rats does not do so in male C3H mice.