A new method for the determination of kinetic and inhibition parameters of cytochromes P450 reactions by means of on-line CE was developed. It is based on transverse diffusion of laminar flow profiles methodology introduced by Krylov et al. that injection procedure was modified. The solutions of an enzyme and its substrates are injected by hydrodynamic pressure as a series of repeated consecutive plugs. Proposed injection of three plugs of enzyme surrounded with plugs of substrates represents a certain trade-off to obtain the reaction mixture with the satisfying homogeneity by the short-injection procedure as possible. Mathematical modeling confirmed the assumption of a consistent distribution of reactants in the final reaction mixture. Kinetic and inhibition studies of cytochrome P450 isoform 2C9's reaction with diclofenac as a probe substrate and sulfaphenazole as a probe inhibitor were conducted in order to prove the practical applicability of the proposed method for on-line screenings of drug metabolism mediated by cytochrome P450 enzymes. As a result, an apparent Michaelis constant of 2.66 +/- 0.18 M, apparent maximum reaction velocity of 7.91 +/- 0.22 nmol min(-1) nmol(-1), Hill coefficient of 1.59 +/- 0.16, half maximal inhibitory concentration of 0.94 +/- 0.04 M and apparent inhibition constant of 0.39 +/- 0.07 M were determined. All these values are in agreement with literature data obtained using different techniques. In addition, less than 30 nL of cytochrome P450 2C9 solution was consumed per analysis in the kinetic and inhibition studies using this method.