Terpenoids play an important role in defense against insects and pathogens in cotton. These terpenoids contain phenolic groups. Metabolites in which the phenolic group has been converted to a methoxy group are less toxic to most insects and pathogens and thus may alter resistance. Here is reported the cloning of a gene from Gossypium barbadense that encodes the enzyme that methylates the phenolic group of desoxyhemigossypol (dHG) exclusively at the 6-position, dHG-6-O-methyltransferase (dHG-6-OMT). Partial peptide sequences from digests of purified dHG-6-OMT were used to design primers for RT-PCR amplification of cDNA fragments from poly(A) mRNA. Fragments were extended to full length using 5' and 3' RACE. The resulting cDNA codes for a 365-residue polypeptide with a calculated molecular weight of 40.6 kDa, in agreement with the molecular mass of purified dHG-6-OMT. When expressed in Escherichia coli, the bacterial lysates showed a high specificity for the methylation of desoxyhemigossypol, differentiating the cloned gene from other pathogen-induced methyltransferases.