1. N-Hydroxylation of aniline and 4-chloroaniline was quantified in rainbow trout microsomal preparations using h.p.l.c.-liquid scintillation methods. Radioactive phenylhydroxylamine and 4-chlorophenylhydroxylamine metabolites were identified by co-elution with non-labelled standards. The method provided resolution of metabolite standards, and quantification of both N-hydroxylated metabolites was achieved without derivatization. 2. The maximum velocities at 25°C were 33.8±1.40 and 22.0±0.98 pmol/min per mg for aniline and 4-chloroaniline N-hydroxylation, respectively. The Km values were 1.0±0.11 and 0.8±0.11 mM for aniline and 4-chloroaniline N-hydroxylation, respectively. These activities were not induced by treatment of the trout with Aroclor 1254 under the conditions of this study. 3. When incubations were performed at 11°C, the physiological temperature of rainbow trout in this study, the Vmax for 4-chloroaniline N-hydroxylation decreased from 22.0 to 6.4 pmol/min per mg and the Km decreased from 0.8 to 0.5 mM. 4. The pH optimum for 4-chloroaniline N-hydroxylation was 8.0 while the pH optimum for aniline N-hydroxylation ranged from 7.4 to 8.0, suggesting the possible contribution of different isoenzymes. 5. The demonstration of aniline and 4-chloroaniline N-hydroxylation by rainbow trout microsomes provides further insight into the high acute: subchronic toxicity ratios observed in fish exposed to these compounds. © 1991 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.