A highly sensitive, simple assay for the determination of 4-nitrocatechol formed during the P450-dependent hydroxylation of p-nitrophenol has been developed. The assay utilizes high-performance liquid chromatography with electrochemical detection. The procedure consists of enzymatic reaction, quenching with trifluoroacetic acid, precipitation of the protein by centrifugation, and direct injection of supernatant aliquots for analysis on a reverse-phase C18 column. The electrochemical detection makes it possible to detect 0.5 pmol of 4-nitrocatechol injected, 30 times less than previously reported for an ultraviolet detector. The detector response was linear with time and with protein content, down to as little as 1.0 microgram of microsomal protein. The usefulness of the method was demonstrated with hepatic microsomal as well as S-9 fractions from both ethanol-treated and control rats, and the dependency of the reaction on cytochrome P450 2E1 activity was proven by immunoinhibition experiments.