Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin-type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O-prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His8-FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. KM values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 μM, respectively. Average turnover number (kcat) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s−1. FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13-dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.