The putative gene fgaMT was identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus. The coding region of fgaMT was amplified by PCR from a cDNA library, cloned into pQE60, and overexpressed in Escherichia coli. FgaMT comprises 339 amino acids with a molecular mass of about 38.1 kDa. The soluble dimeric His(6)-FgaMT was purified to near homogeneity and characterized biochemically. FgaMT was found to catalyze the N-methylation of 4-dimethylallyltryptophan in the presence of S-adenosylmethionine, resulting in the formation of 4-dimethylallyl-l-abrine, which was identified by NMR and mass spectrometry analysis. Therefore, FgaMT represents the second pathway-specific enzyme in the biosynthesis of ergot alkaloids. The enzyme did not require metal ions for its enzymatic reaction and showed a relatively high specificity toward the prenyl moiety at position C-4 of the indole ring. 4-Dimethylallyltryptophan derivatives with modification at the indole ring were also accepted by FgaMT as substrates. K(m) values for 4-dimethylallyltryptophan and S-adenosylmethionine were determined at 0.12 and 2.4 mm, respectively. The turnover number was 2.0 s(-1).