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. The maximum velocities at 25 degrees 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. When incubations were performed at 11 degrees 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. 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. 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.