Microsomal 4-methyl sterol oxidase of cholesterol biosynthesis from lanosterol has been assayed to date by coupling of the NAD(P)H-dependent oxygenase to a NAD-dependent decarboxylase in a two-step incubation procedure. A simple assay of 4-methyl sterol oxidase of rat liver microsomes has now been developed with the model substrate, 4-hydroxyp4C]methylene-5a-cholest-7-en-3-one. In the presence of oxygen and NADPH, the 30-~4C-model substrate is oxidized directly to a 3-ketosteroid and ~4CO2, which is collected and counted. Conditions for measurement of initial rates of oxidation of the model substrate have been established. With the model substrate, the maximal velocity is four-to five-fold greater than the rate observed with 4a-methyl sterol substrates. Furthermore, when methyl sterol oxidase is measured with both the one-step and two-step assays, parallel effects are produced upon addition of either competitive or noncompetitive inhibitors in vitro; similarly, oxidative attack on each substrate is enhanced equally when rats are treatedin vivo with a bile salt sequestrant. Thus, the direct one-step assay of oxidase activity with the model 4-hydroxy["C]methylene sterol substrate is rapid, the observed rates are rapid, and the enzymic steps in the multienzymic, mixed function oxidase may be elucidated with this simplified procedure.