We investigated the in vitro metabolism by mouse and rat liver 9000 x g supernatant of the strong tumor initiator, 5,11-dimethyichrysene (5,11-diMeC) and its inactive analogue, 5,12-dimethylchrysene (5,12-diMeC). Ethyl acetate soluble metabolites were separated by h.p.l.c. and identified by their u.v. and m.s. and by comparison to selected synthetic reference standards. Both compounds were converted to dihydrodiols, chrysenols, hydroxymethylchrysenes, and hydroxymethylchrysenols. The peri 12-methyl group of 5,12-diMeC strongly inhibited metabolism at the adjacent 1,2-positions. Thus, the ratio of 7-hydroxy-5,12-diMeC to 1-hydroxy-5,12-diMeC was ∼100 to 1 when 5,12-diMeC was metabolized by liver supernatants from 3-methyicholanthrene pretreated mice and rats. In addition, 7,8-dihydro-7,8-dihydroxy-5,12-diMeC was preferentially formed over 1,2-dihydro-1,2-dihydroxy-5,12-diMeC by liver supernatants from control, 3-methylcholanthrene pretreated, and Aroclor pretreated animals. In contrast, the presence of a methyl group at the 11 position of 5,11-diMeC did not inhibit formation of 1-hydroxy-5,11-diMeC or 1,2-dihydro-1,2-dihydroxy-5,11-diMeC. Since 1,2-dihydro-1,2-dihydroxy metabolites of 5-methylchrysene derivatives are potential proximate tumorigens, these results may provide a basis for the higher tumorigenicity of 5,11-diMeC than of 5,12-diMeC.