There is a direct relationship between the metabolism and mutagenicity of N-nitrosohexamethyleneimine (NO-HEX) in the presence of uninduced and AC- and PB-induced S8 and S9 fractions from rats and hamsters. Although alpha-hydroxylation is the most important process in the formation of mutagens, NO-HEX may be hydroxylated on the beta- and gamma-carbon atoms as well. beta- and gamma-hydroxyNO-HEX do not appear to play a significant role in the total mutagenicity of NO-HEX. Using rat liver subcellular fractions, beta- and gamma-hydroxyNO-HEX are only marginally mutagenic compared with NO-HEX. With hamster S9 fractions, beta-hydroxyNO-HEX is equally as mutagenic as NO-HEX itself, but gamma-hydroxyNO-HEX is a much less potent mutagen. However, beta-hydroxyNO-HEX is produced in small amounts and therefore does not contribute greatly to the total mutagenicity of NO-HEX.