A strategy based on the random isolation and screening of soybean cDNAs encoding cytochrome P450 monooxygenases (P450s) was used in an attempt to identify P450 isozymes involved in herbicide metabolism. Nine full-length (or near-full-length) P450 cDNAs representing eight distinct P450 families were isolated by using PCR-based technologies. Five of the soybean P450 cDNAs were expressed successfully in yeast, and microsomal fractions generated from these strains were tested for their potential to catalyze the metabolism of 10 herbicides and 1 insecticide. In vitro enzyme assays showed that the gene product of one heterologously expressed P450 cDNA (CYP71A10) specifically catalyzed the metabolism of phenylurea herbicides, converting four herbicides of this class (f luometuron, linuron, chlortoluron, and diuron) into more polar compounds. Analyses of the metabolites suggest that the CYP71A10 encoded enzyme functions primarily as an N-demethylase with regard to f luometuron, linuron, and diuron, and as a ring-methyl hydroxylase when chlortoluron is the substrate. In vivo assays using excised leaves demonstrated that all four herbicides were more readily metabolized in CYP71A10-transformed tobacco compared with control plants. For linuron and chlortoluron, CYP71A10-mediated herbicide metabolism resulted in significantly enhanced tolerance to these compounds in the transgenic plants.Cytochrome P450 monooxygenases (P450s) are ubiquitous hemoproteins present in microorganisms, plants, and animals. Composed of a large and diverse group of isozymes, P450s mediate a great array of oxidative reactions. Most of these activities support biosynthetic processes such as phenylpropanoid, fatty acid, and terpenoid biosynthesis. Others metabolize natural products (e.g., the monoterpene camphor) or play a role in xenobiotic detoxification (1). In a typical P450-catalyzed reaction, one atom of molecular oxygen (O 2 ) is incorporated into the substrate, and the other atom is reduced to water by using NADPH. For most eukaryotic P450s, NADPH͞cytochrome P450 reductase, a membrane-bound flavoprotein, transfers the necessary two electrons from NADPH to the P450 (2).After the initial report of Frear et al. (3), who demonstrated the metabolism of monuron by a mixed-function oxidase located in a microsomal fraction of cotton seedlings, an extensive body of evidence has accumulated supporting the involvement of P450s in the metabolism and detoxification of numerous herbicides (1, 2). Differential herbicide-metabolizing P450 activities are believed to represent one of the mechanisms that enables certain crop species to be more tolerant of a particular herbicide than other crop or weedy species. Although the association of P450s with herbicide metabolism has been investigated for several decades, reports on the successful cloning and expression of P450 cDNAs encoding herbicide-metabolizing isozymes are relatively recent. In one example, constructs using the rat CYP1A1 cDNA conferred chlortoluron resistance in tobacco by enhancing herbicid...