Fluorescent pseudomonads produce yellow-green siderophores when grown under conditions of iron starvation. Here, the characterization of the pvdF gene, which is required for synthesis of the siderophore pyoverdine by Pseudomonas aeruginosa strain PAO1, is described. A P. aeruginosa pvdF mutant was constructed and found to be defective for production of pyoverdine, demonstrating the involvement of PvdF in pyoverdine synthesis. Transcription analysis showed that expression of pvdF was regulated by the amount of iron in the growth medium, consistent with its role in siderophore production. DNA sequencing showed that pvdF gives rise to a protein of 31 kDa that has similarity with glycinamide ribonucleotide transformylase (GART) enzymes involved in purine synthesis from a wide range of eukaryotic and prokaryotic species. Chemical analyses of extracts from wild-type and pvdF mutant bacteria indicated that the PvdF enzyme catalyses the formylation of N(5)-hydroxyornithine to give rise to N(5)-formyl-N(5)-hydroxyornithine, a component of pyoverdine. These studies enhance understanding of the enzymology of pyoverdine synthesis, and to the best of the authors' knowledge provide the first example of involvement of a GART-type enzyme in synthesis of a secondary metabolite.