Bordetella pertussis and Bordetella bronchiseptica, gram-negative respiratory pathogens of mammals, possess a heme iron utilization system encoded by the bhuRSTUV genes. Preliminary evidence suggested that expression of the BhuR heme receptor was stimulated by the presence of heme under iron-limiting conditions. The hurIR (heme uptake regulator) genes were previously identified upstream of the bhuRSTUV gene cluster and are predicted to encode homologs of members of the iron starvation subfamily of extracytoplasmic function (ECF) regulators. In this study, B. pertussis and B. bronchiseptica ΔhurI mutants, predicted to lack an ECF σ factor, were constructed and found to be deficient in the utilization of hemin and hemoglobin. Genetic complementation of ΔhurI strains with plasmid-borne hurI restored wild-type levels of heme utilization. B. bronchiseptica ΔhurI mutant BRM23 was defective in heme-responsive production of the BhuR heme receptor; hurI in trans restored heme-inducible BhuR expression to the mutant and resulted in BhuR overproduction. Transcriptional analyses with bhuR-lacZ fusion plasmids confirmed that bhuR transcription was activated in iron-starved cells in response to heme compounds. Heme-responsive bhuR transcription was not observed in mutant BRM23, indicating that hurI is required for positive regulation of bhu gene expression. Furthermore, bhuR was required for heme-inducible bhu gene activation, supporting the hypothesis that positive regulation of bhuRSTUV occurs by a surface signaling mechanism involving the heme-iron receptor BhuR.