A high-pressure liquid chromatographic assay for alpha hydroxylation of N-nitrosopyrrolidine by isolated hepatic microsomes was developed. Mixtures consisting of N-nitrosopyrrolidine, microsomes, and an NADPH-generating system were incubated at 37 degrees. The major product of alpha hydroxylation of N-nitrosopyrrolidine, 2-hydroxytetrahydrofuran, was trapped by the addition of 2,4-dinitrophenylhydrazine reagent to form 4-hydroxybutyraldehyde-2,4-dinitrophenylhydrazone. The latter was quantified by reverse-phase high-pressure liquid chromatography. Under optimal conditions, as determined by varying protein and substrate concentrations, the alpha hydroxylation of N-nitrosopyrrolidine was linear for at least 90 min and showed characteristics typical of the microsomal mixed-function oxidase system, such as inhibition by CO and induction by pretreatment of male F-344 rats with Aroclor. N-Nitrosopyrrolidine exhibited type II spectral changes upon interaction with isolated hepatic microsomes. A close correspondence between binding affinity and alpha hydroxylation of N-nitrosopyrrolidine was observed.