Chapters 10-20 describe assay methods for determining enzyme activities that are frequently employed as markers for specific cytochrome P450 (P450) enzymes in human tissues. However, under certain circumstances, e.g., when the objective is to verify that individual cDNA-expressed P450 enzymes are expressed in catalytically active form, it is often advantageous to assay the panel of enzymes using a “general” P450 substrate, one that is metabolized by multiple P450 enzymes. An example of a general P4.50 substrate is 7-ethoxycoumarin. Enzyme kinetic analysis with human liver microsomes indicates that 7-ethoxycoumarin is metabolized by at least two P450 enzymes or by two groups of P450 enzymes that are kinetically distmguishable (1 , 2 ). The apparent Km and maximal specific activity (i.e., specific activity at saturating substrate concentration) values for 7-ethoxycoumarin O-deethylation by human liver microsomes are 11–27 ?M and 0.05–0.21 nmol/min/mg microsoma1 protein, respectively, for the low Km component. By contrast, for the high Km component, the apparent Km is approx 150 ?M and the maximal specific activity is 0.35–0.95 nmol/min/mg microsomal protein (2 ). Experiments with recombinant human P450s have shown that multiple enzymes in the human CYP1, CYP2, and CYP3 families are active catalysts of 7-ethoxycoumarin O-deethylation (3 ). Consequently, the 7-ethoxycoumarin O-deethylase assay has been used to verify the catalytic activity of a panel of recombinant human P450s in the CYPl, CYP2 and CYP3 families (4 –6 ).