Iron acquisition by the gram-negative pathogens Bordetella bronchiseptica and Bordetella pertussis is thought to occur by hydroxamate siderophore-mediated transport as well as an apparently siderophore-independent process by which host transferrins bind to bacterial surface receptors. We constructed B. bronchiseptica mutants deficient in siderophore activity by insertional mutagenesis with miniTn5/lacZ1. The mutants could be placed into four distinct complementation groups, as determined from cross-feeding assays which demonstrated restored siderophore synthesis. Mutants deficient in siderophore activity were BRM1, BRM6, and BRM9, exhibiting approximately 36 to 41% of wild-type siderophore levels, and BRM3 and BRM8, which appeared to produce very little or no detectable siderophore. Mutant BRM4 was found to be a leucine auxotroph, while mutants BRM2 and BRM7 could synthesize siderophore only in low-iron medium which was supplemented with various amino acids. Evaluation of all transcriptional fusions revealed an apparent lack of iron-regulated lacZ expression. Genomic regions flanking the transposable element in the siderophore mutants were homologous with B. pertussis chromosomal DNA, while bioassays suggested siderophore cross-feeding between B. pertussis and B. bronchiseptica. These results indicate probable similarity between the siderophore biosynthetic and transport systems of the two species.