In two separate in vivo studies, ethionine was evaluated for carcinogenic activity in mice. In the first study, DL-ethionine was fed in a chow diet at 0 (controls), 0.1 (low dose, LD) and 0.25% (high dose, HD) concentrations to the following groups of mice (30 animals/group): Swiss Webster CD-1 females, BALB/c males, and C3H/HeN males and females. Because of severe toxicity, BALB/c females were fed 0.05% (LD) and 0.1% (HD) ethionine. The Swiss and BALB/c mice were maintained on their respective diets for up to 105 weeks before killing whereas the C3H mice were killed at 68 weeks because of the high spontaneous incidence of liver tumors in this strain. The percentages of animals at risk (surviving the time to the first liver tumor recorded in each sex and strain) that bore liver tumors were as follows: Swiss female control, 0% (0/29), Swiss female LD, 87% (20/23); Swiss female HD, 89% (16/18); C3H male controls, 35% (8/23); C3H male LD, 55% (16/29); C3H male HD, 58% (15/26); C3H female controls, 5% (1/20); C3H female LD, 60% (12/20); C3H female HD, 92% (12/13); BALB/c male controls, 4% (1/23); BALB/c male LD, 8% (2/24); BALB/c male HD, 31% (5/16); BALB/c female controls, 0% (0/30); BALB/c female LD, 52% (14/27); and BALB/c female HD, 92% (12/13). The female mice were more responsive than the males in developing liver tumors. The results of the feeding study are compared with those obtained in a second study in which C3H female mice were intubated with 0, 150 or 500 mg DL-ethionine/kg body wt three times per week for 30 weeks and killed at 2 years. Only the LD mice showed a significantly increased incidence of liver tumors (20/39) as compared to controls (12/41) or HD mice (7/37) in the latter study. The hepatic levels of the major ethionine metabolite and methylase inhibitor, S-adenosylethionine (AdoEt), as well as of the endogenous methyl group donor, S-adenosylmethionine (AdoMet) were determined in Swiss female mice fed either 0.1 or 0.3% in the diet for 1-6 weeks. Hepatic AdoEt levels ranged from 37 to 80 micrograms/g liver in the LD animals and from 61 to 203 micrograms/g liver in the HD group; levels of the endogenous metabolite AdoMet correspondingly dropped to 65% of the normal levels. The present results (i) extend to different strains and to both sexes previous observations demonstrating the hepatocarcinogenic activity of ethionine in mice; and (ii) indicate that as in the rat such activity may be exerted through the formation of AdoEt.