The linkage of isoprenoid and aromatic moieties, catalyzed by aromatic prenyltransferases (PTases), leads to an impressive diversity of primary and secondary metabolites, including important pharmaceuticals and toxins. A few years ago, a hydroxynaphthalene PTase, NphB, featuring a novel ten-stranded β-barrel fold was identified in Streptomyces sp. strain CL190. This fold, termed the PT-barrel, is formed of five tandem ααββ structural repeats and remained exclusive to the NphB family until its recent discovery in the DMATS family of indole PTases. Members of these two families exist only in fungi and bacteria, and all of them appear to catalyze the prenylation of aromatic substrates involved in secondary metabolism. Sequence comparisons using PSI-BLAST do not yield matches between these two families, suggesting that they may have converged upon the same fold independently. However, we now provide evidence for a common ancestry for the NphB and DMATS families of PTases. We also identify sequence repeats that coincide with the structural repeats in proteins belonging to these two families. Therefore we propose that the PT-barrel arose by amplification of an ancestral ααββ module. In view of their homology and their similarities in structure and function, we propose to group the NphB and DMATS families together into a single superfamily, the PT-barrel superfamily.