<jats:title>ABSTRACT</jats:title> <jats:p> Mutants of <jats:italic>Pseudomonas fluorescens</jats:italic> WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb <jats:italic>Eco</jats:italic> RI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of <jats:italic>P. putida</jats:italic> . Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs ( <jats:italic>pmsC</jats:italic> and <jats:italic>pmsB</jats:italic> ) showed homologies with chorismate-utilizing enzymes; a third ORF ( <jats:italic>pmsE</jats:italic> ) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene ( <jats:italic>pmsA</jats:italic> ). A putative promoter region and two predicted iron boxes were localized upstream of <jats:italic>pmsC</jats:italic> . We determined by reverse transcriptase-mediated PCR that the <jats:italic>pmsCEAB</jats:italic> genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in <jats:italic>P. putida</jats:italic> and <jats:italic>Escherichia coli</jats:italic> cells. In <jats:italic>E. coli</jats:italic> , deletions affecting the first ORF ( <jats:italic>pmsC</jats:italic> ) diminished SA production, whereas deletion of <jats:italic>pmsB</jats:italic> abolished it completely. The <jats:italic>pmsB</jats:italic> gene induced low levels of SA production in <jats:italic>E. coli</jats:italic> when expressed under control of the <jats:italic>lacZ</jats:italic> promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn <jats:italic>5</jats:italic> mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.