Twelve kinds of alkylated derivatives of 4-nitroquinoline 1-oxide (4NQO) were tested for their mutagenicity on S. typhimurium TA100. The assay method was slightly modified from the original procedure of Ames in order to obtain a better correlation with intrinsic chemical properties of the compounds. It was found that mutagenic potencies of these derivatives were linearly correlated with the metabolic rates of 4NQO's to the corresponding 4-hydroxyaminoquinoline 1-oxides (4HAQO's), the correlation coefficient being 0.930. The steric requirements for enzymic reduction of the nitro group seem to be the major determinant of the mutagenic activity of the derivatives, and surprisingly, the substituents do not have any appreciable effect either on the subsequent metabolic activation step (aminoacylation of 4HAQO's) or on the ultimate nonenzymatic modification step.