For exploration of the factors affecting dimethylnitrosamine (DMN) mutagenicity, for gathering of information on the metabolism of DMN, a frequently used and relatively well-understood carcinogen, and for explanation of metabolic variations in DMN carcinogenicity, parallel in vitro assays of the microsomal activation of DMN to a mutagen and of DMN demethylation were performed. Salmonella typhimurium G46 reversions to histidine independence increase linearly with time of incubation for 30 min. At low concentrations of microsomal protein, increases in protein yield a more than proportional increase in mutations. Increasing DMN concentration saturates the enzyme, yielding less demethylation and fewer mutations proportionately. Mutagenesis is completely inhibited by 1 mM 2-diethyl-aminoethyl-2,2-diphenylvalerate. When both DMN and microsomal protein are varied at high concentrations, there is a simple linear relationship between mutagenicity and DMN demethylase activity. Thus DMN demethylase activity may be the primary controlling factor in the metabolism of DMN to a mutagen, and probably to a carcinogen; other simultaneous pathways of DMN metabolism proportional to demethylation have not been ruled out. Induction with both phenobarbital and 3-methylcholanthrene (3-MC) increased rat and mouse liver DMN demethylase activity. Mouse liver microsomes from the C57BL/6 strain demethylate DMN at a markedly lower rate than do microsomes from the C3H strain, but after 3-MC induction the relationship is reversed. Strain differences in activation of DMN were not found in the activation of diethylnitrosamine to a mutagen. Hepatic dealkylation of DMN and diethylnitrosamine to active mutagenic metabolites is increased in both rats and mice by both 3-MC and phenobarbital induction, which is in contrast to the findings of others that 3-MC and phenobarbital induction, which is in contrast to the findings of others that 3-MC decreases the incidence of DMN-induced hepatic tumors in rats, and phenobarbital decreases the incidence of diethylnitrosamine-induced hepatic tumors in mice.