Rainbow trout liver microsomes were used to study the O-demethylation and ring hydroxylation of 4-methoxyphenol (4-MP) (4-hydroxyanisole) at 11 and 25°C by directly measuring the production of the primary metabolite hydroquinone (HQ), 4-methoxycatechol (4-MCAT), and additional metabolites. An HPLC method with integrated ultraviolet (UV) and electrochemical detection (ECD) was developed for metabolite identification and quantification at low concentrations. Sample handling with appropriate buffers, solvents, low temperature and light prevented loss of extremely labile metabolites. Saturation kinetics for the production of HQ via O-demethylation of 4-MP (0.66–40 mM) was never achieved, with substrate solubility being the limiting factor. The linear rate of HQ formation at 11°C was 22.0±2.2 (coefficient±S.E., r2=0.91) pmol min−1 per mg protein per mM substrate, and at 25°C was 34.0±1.3 (r2=0.99) pmol min−1 per mg protein per mM substrate. The second major microsomal metabolite 4-MCAT was also identified, with linear rates of ring hydroxylation determined to be 19.0±1.6 (r2=0.94) and 17.2±0.5 (r2=0.99) pmol min−1 per mg protein per mM substrate at 11 and 25°C, respectively. Unlike HQ production, the rate of 4-MCAT production was found to be similar at the two temperatures when linear formation rates were corrected for the effect of temperature on substrate and product solubility at 11°C. Measurement of `freely dissolved' fraction was essential to the accurate determination of ring hydroxylation and O-demethylation reaction rates in rainbow trout microsomes incubated at physiological temperature. Experimental conditions were shown to affect dissolved 4-MP and HQ at 11°C (verified using microdialysis) while not altering substrate and product levels at 25°C. Small but detectable levels of 1,4-benzoquinone were observed in 4-MP microsomal incubations. 1,2,4-Trihydroxybenzene was also detected, with possible routes of production through hydroxylation of HQ or O-demethylation of 4-MCAT. A metabolic scheme for bioactivation of 4-MP is proposed and the significance of observed metabolic conversions in rainbow trout microsomes discussed in relation to aquatic toxicity of 4-MP.