Pseudobactin 358 is the yellow-green fluorescent siderophore produced by Pseudomonas putida WCS358 in conditions of iron limitation. The genes encoding for siderophore biosynthesis are iron-regulated at the transcriptional level. Previous work has shown that a positive regulator, PfrA, is absolutely required for the activation under iron-limiting conditions of pseudobactin 358 biosynthesis. In this study we identified a set of Tn5 insertion mutants of strain WCS358 which lost the ability to activate an iron-regulated siderophore promoter. These mutants no longer produced pseudobactin 358. Molecular analysis revealed that they carried a Tn5 insertion in a gene, designated pfrl (Pseudomonas ferric regulator), which codes for a protein (Pfrl) of 19.5 kDa. Pfrl contains a putative helix-turn-helix motif typical of DNA-binding proteins and has homology to two DNA-binding transcriptional activators, Fecl from Escherichia coli and Pupl from P. putida. The proposed role of Pfrl in strain WCS358 is an activator protein regulating pseudobactin 358 biosynthesis under iron limitation. The pfrl promoter region contains a sequence which displays high identity to the Fur-box consensus. This 19 bp consensus sequence is recognized by Fur, an iron-binding repressor protein found in many different bacteria. The E. coli Fur protein can bind to the pfrl promoter region, indicating that this activator gene is likely to be iron-regulated by Fur. We also report the identification and characterization of the P. putida WCS358 fur gene. The Fur protein of strain WCS358 is structurally and functionally similar to other cloned Fur proteins from other bacterial species.