A method is described for the qualitative and quantitative determination of the isoenzymes of cytochrome P-450 from rat liver microsomes. Microsomes are incubated with the endogenous steroid 17 beta-testosterone, which results in the formation of a number of stereo-specific hydroxylation products of testosterone. The hydroxylated products were identified using standards or by comparison with data from the literature. The products can be analysed by reversed-phase gradient high-performance liquid chromatography. The assay has been optimised for pH, linearity and time of incubation. An evaluation of the assay was performed for different kinds of microsomes, microsomal dilution and specificity for particular cytochrome P-450 isoenzymes.