1. We evaluated the specificity of 15 substrates and 14 inhibitors of the cytochrome P450s using nine human P450 forms expressed in HepG2 cells using a recombinant vaccinia virus and also in human liver microsomes. 2. Coumarin, 7-ethoxyresorufin, 7-benzyloxyresorufin, tolbutamide, aniline and diazepam were form-selective substrates towards CYP2A6, the CYP1A subfamily, CYP2B6, the CYP2C subfamily, CYP2E1 and the CYP3A subfamily respectively. However, a selective substrate for CYP2D6 was not found among the chemicals tested. 3. SKF-525A inhibited > 40% of the metabolic activity of all substrates tested, and the inhibitory effects differed among P450 forms. Sulphaphenazole, 7,8-benzoflavone, quinidine and troleandomycin were selective inhibitors of the CYP2C subfamily (except CYP2C19), the CYP1A subfamily, CYP2D6 and the CYP3A subfamily respectively. Methoxsalen (CYP2A6 inhibitor) inhibited the metabolic activity of CYP1A2 as well as that of CYP2A6. Diethyldithiocarbamate (CYP2E1 inhibitor) inhibited the metabolic activities of CYP2A6 and CYP2C19 in addition to that of CYP2E1. 4. Our results indicated that substrates and inhibitors reported as P450 selective probes are not necessarily specific for individual human P450 forms. These results may provide useful information regarding human P450 substrates and inhibitors in vitro using human liver microsomal samples.