Siderophores are high-affinity iron-chelating compounds produced by bacteria for iron uptake that can act as important virulence determinants for both plant and animal pathogens. Genome sequencing of the plant pathogen, Streptomyces scabies 87-22, revealed the presence of a putative pyochelin biosynthetic gene cluster (PBGC). Liquid chromatography-mass spectrometry (LC-MS) analyses of culture supernatants of S. scabies mutants in which expression of the cluster is upregulated and which lack a key biosynthetic gene from the cluster indicated that pyochelin is a product of the PBGC. HPLC comparisons with authentic standards on a homochiral stationary phase confirmed that pyochelin and not enantio-pyochelin (ent-pyochelin) is produced by S. scabies. Transcription of the S. scabies PBGC occurs via ~19 kb and ~3 kb operons and transcription of the ~19 kb operon is regulated by TetR- and AfsR-family proteins encoded by the cluster. This is the first report of pyochelin production by a Gram-positive bacterium; interestingly regulation of pyochelin production is distinct from characterized pyochelin biosynthetic gene clusters in Gram-negative bacteria. Though pyochelin-mediated iron acquisition by Pseudomonas aeruginosa is important for virulence, in planta bioassays failed to demonstrate that pyochelin is required for development of disease symptoms on excised potato tuber tissue or radish seedlings.