Kinetics of testosterone 6beta-hydroxylation were determined using a reconstituted system that consisted of CYP3A4, cytochrome b5 and NADPH-cytochrome P450 oxidoreductase (OR) with similar ratios as those seen in human liver microsomes and compared with those determined using human liver microsomes. Two reconstituted systems were constructed in accordance with two human liver microsomal samples that showed extremely high and low ratios of OR/CYP3A4. The Km values of testosterone 6beta-hydroxylation obtained from the reconstituted systems with high and low OR/CYP3A4 ratios were 29.3 and 35.2 microM, respectively, which were similar to that of the corresponding human liver microsomal samples (23.2 and 40.0 microM, respectively). However, Vmax values obtained from the reconstituted systems (3.7 and 0.8 pmol/min/pmol CYP3A4) were much lower than those from the human liver microsomes (44.2 and 31.1 pmol/min/pmol CYP3A4). The results suggest that the interaction between substrate and CYP3A4 in the reconstituted systems appear to be similar to human liver microsomes but that the velocity of the substrate metabolism in the reconstituted systems is different from that in human liver microsomes. In conclusion, our reconstituted systems could be used for the determination of affinity but not for the determination of the maximum velocity of substrate metabolism. Further studies on the protein-protein interactions between CYP3A4, OR, cytochrome b5 and/or a specific lipid environment are required to establish a reconstituted system showing similar kinetic properties to those of human liver microsomes.