Using a microtiter plate (MTP) assay consists of recombinant cytochromes P450 and fluorescent probes, we evaluated inhibitory effects of commercially available model-compounds, 18 typical substrates and 8 selective inhibitors, on nine cytochromes P450 (CYPs) activities. The IC(50) values obtained from the assay were used to estimate inhibition constant (Ki) values, assuming competitive inhibition. The Ki values calculated from IC(50) (the Ki(-cal)) with the MTP assay using recombinant CYPs were compared with the Ki values (the Ki(-rep)), reported for human liver microsomes (HLM). Regarding all the inhibitory effects of the 26 test compounds on each CYP activity, a good correlation (r(2)=0.7306) was found between Ki(-cal) and Ki(-rep). The inhibitory patterns of some compounds on the five major CYP isoforms were estimated, using the MTP assay with the preincubation method. Furafylline and erythromycin, both mechanism based inhibitors, strongly inhibited CYP1A2 and CYP3A4 activity, respectively and their inhibitory effects increased depending on the preincubation time. In contrast, the inhibitory effects of phenacetin, diclofenac, S-mephenytoin, dextromethorphan, bufuralol and terfenadine, typical substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, respectively, on each recombinant CYP activity decreased after preincubation. Therefore, the MTP assay is a useful high throughput screening method to evaluate inhibitory effects of new drug candidates on 9 CYP isoforms in HLM. In addition, the MTP assay with the preincubation method might be beneficial to estimate inhibitory patterns on CYP isoforms of new drug candidates and to estimate main CYP isoforms responsible for metabolism of these compounds.