Nicotine in tobacco may be demethylated to form nornicotine, a precursor to N'-nitrosonornicotine which is one of the tobacco specific nitrosamines formed by microbial action during curing and subsequent leaf storage and processing. We employed a PCR-based strategy using degenerate primers designed on cytochrome P450-conserved regions to clone numerous P450 fragments and full-length P450 genes from tobacco leaves in which nicotine demethylase had been induced by ethylene treatment. From microarray hybridizations using ethylene-treated converter and nonconverter samples as probe sources, we identified two full-length genes and one gene fragment that were highly expressed in the converter materials. The fragment was a partial sequence of both full-length genes which had 99.8% sequence homology. The two full-length genes and two additional P450 genes with similar homology were expressed in yeast. Microsomal preparations from yeast harboring one gene, D121-AA8, showed significant demethylation of radio-labeled nicotine while microsomes from yeast cells containing the remaining three genes had no activity. Demethylation activity of yeast expressed D121-AA8 was decreased significantly with inhibitors specific for P450 enzymes. Results from DNA sequence analyses and enzyme assays with P450 inhibitors suggest strongly that the cloned nicotine demethylase gene is a cytochrome P450 enzyme.