Two enzymic forms, with different kinetic characteristics and responding in opposite ways to in vivo “enzyme inducer” pretreatment, underlie hepatic dimethylnitrosamine(DMN)-demethylase activity. Determination of the Hofstee plot of DMN-demethylase using a DMN substrate concentration range of 0.5 to 200 mM yields three intersecting line segments from which widely different Km and Vmax values may be calculated. Identically patterned Hofstee plots are obtained with rat and mouse postmitochondrial supernatant fractions, as well as with the isolated microsomes, yielding for the respective segments, similar Km values. The low-substrate-range line segment (0–4 mM) and the high-substrate-range line segment (50–200 mM) correspond, in both the rat and the mouse, to two different enzymic forms of DMN-demethylase (DMN-demethylase I and II, respectively) which have different regulatory characteristics: (A) Pretreatment of rats and mice with the polychlorinated biphenyl, Aroclor 1254, brings about repression of DMN-demethylase I (determined at 4 mM DMN) and induction of DMN-demethylase II (determined at 200 mM DMN); both responses are stronger in the rat than in the mouse. Kinetic experiments show that the differential responses of the two DMN-demethylases to Aroclor pretreatment are paralleled by corresponding changes in the Vmax values. (B) Pretreatment with phenobarbital represses enzyme I in both species; however, it induces enzyme II only in the rat. (C) Pretreatment with 3-methylcholanthrene substantially represses DMN-demethylase I in the rat; however, it is ineffective to significantly influence the activity of enzymic form II in the rat and of either enzymic form in the mouse. There is no evidence that the intermediate line segment would correspond to an additional enzymic form. The genetically distinct nature of the two forms of hepatic DMN-demethylase is suggested by the substantial differences in the strain-dependence of the repressibility of enzyme I and inducibility of enzyme II in a series of 9 inbred strains of mice. The mouse lung contains only the inducible-type of DMN-demethylase; however, the ranking of inducibilities is different from the hepatic enzyme II. Beyond about 300 mM further increase of DMN concentration in the assay system brings about progressive decrease of DMN-demethylase activity owing probably to the protein denaturing ability of DMN at high concentrations.