Flavonol 3-O-galactosyltransferase (F3GalTase) is a pollen-specific enzyme which glycosylates the flavonols required for germination in petunia. The highly restricted tissue-specific expression and substrate usage make F3GalTase unique among all other flavonoid glycosyltransferases (GTs) described to date, including the well characterized Bronze 1 (Bz1) gene of maize. RFLP mapping, DNA gel blot, and sequence analyses showed that the single copy F3galtase gene has a different genomic organization than Bz1. Within the promoter of F3galtase are potential regulatory motifs which may confer pollen-specific gene expression and activation by Myb and bHLH transcription factors. However, we provide evidence that F3galtase is not regulated by An4, which encodes a Myb factor known to regulate anthocyanin accumulation in petunia anthers. An unexpected feature of the F3galtase promoter was the presence of large blocks of chloroplast and mitochondrial DNA. Gel blot analyses of genomic DNA from the progenitors of Petunia hybrida, as well as from Nicotiana tabacum, indicated that migration of organellar DNA into the F3galtase gene was an ancient event that occurred prior to speciation of the Solanaceae. Together with enzyme assays and HPLC analyses of pollen extracts from tobacco, tomato, and potato, these results confirmed that the unique F3galtase gene structure, enzyme activity, and pollen-specific flavonol glucosylgalactosides are conserved throughout the Solanaceae.