An electrophoretically mediated microanalysis method for the determination of CYP3A4 activity using testosterone and nifedipine as substrates was developed. Initially, the enzymatic reaction was performed off-line and the samples were subsequently injected into the capillary by pressure. The CYP3A4 activity was determined by quantitation of the reactant cofactor, NADPH. To further optimize, speed-up and miniaturize the enzyme assay, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product cofactor, NADP, employing the plug-plug mode of electrophoretically mediated microanalysis. An amplification step was introduced by means of an on-capillary incubation of 15 min, in order to accumulate enough reaction product to detect spectrophotometrically at 260 nm. This setup resulted in a fully automated assay, which can be carried out in less than 35 min. Using the Lineweaver-Burk equation, the Michaelis constants (K(m)) for the oxidation of testosterone and nifedipine by CYP3A4 were calculated to be 58.6+/-8.3 and 19.1+/-2.4 muM, respectively, which are consistent with off-line assay and previously reported values.