The in vitro metabolism of N-nitrosohexamethyleneimine by lung and liver microsomes and cytosol from uninduced male Fischer rats is described. Metabolites produced by both organs appeared to be identical. The liver subcellular fractions had a lower Km (0.6 mM) than did lung fractions (3 mM) and metabolized 2.5 to 5 times as much nitrosamine per mg protein. Our results, together with those from our earlier studies, indicate that, as the size of the carbon ring increases from nitrosopyrolidine to nitrosohexamethyleneimine, lung microsomes had an increased affinity for the cyclic nitrosamines; they was only a small effect with liver enzymes. Ths suggests that microsomal enzymes that metabolize cyclic nitrosamines in rat livers and lungs are not the same. The first stable alpha-hydroxylation product, 6-hydroxyhexanal, was not detected in reactions involving microsomes alone. Apparently, this compound is rapidly converted to 1,6-hexanediol by liver or lung microsomes. The presence of cytosol was needed for the full conversion of these metabolites to xi-hydroxycaproate and maximal alpha-hydroxylation activity. xi-Aminocaproate was always found in direct proportion to the hydroxyacid, suggesting that both acids arise from the same alpha-hydroxylation event by different breakdown mechanisms. beta- and gamma-hydroxynitrosohexamethyleneimine were not metabolized significantly by rat liver enzymes and thus, in this species, may be "detoxification products" of N-nitrosohexamethyleneimine.