7-Ethoxycoumarin is metabolized by many cytochrome P450 enzymes active in foreign compound metabolism and has been used as a prototypic substrate to monitor P450 (P450) activity in both hepatic and extrahepatic tissues. A spectrofluorometric method is described for determination of P450-catalyzed 7-ethoxycoumarin O-deethylation. Following acidification of the incubation mixture, the enzymatic product, 7-hydroxycoumarin, is recovered by a double-extraction procedure and measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. This method is applicable to enzymatic studies to determine the catalytic activity of cDNA-expressed human enzymes in the CYP1, CYP2, and CYP3 families, and 7-ethoxycoumarin O-deethylation activity in microsomes isolated from liver and other tissues.