A putative dimethylallyltryptophan synthase gene,fgaPT2, was identified in the genome sequence ofAspergillus fumigatus.fgaPT2was cloned and overexpressed inSaccharomyces cerevisiae. The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convertl-tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD.Kmvalues forl-tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 μM, respectively. Metal ions, such as Mg2+and Ca2+, enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction.