Vibrio anguillarum can utilize heme and hemoglobin as iron sources. Nine genes, huvA, huvZ, huvX, tonB1, exbB1, exbD1, huvB, huvC, huvD, encoding the proteins involved in heme transport and utilization, are clustered in a 10-kb region of chromosomal DNA. Reverse Transcriptase-PCR analysis demonstrated that the gene cluster is arranged into three transcriptional units: (1) huvA, (2) huvXZ, and (3) tonB1exbB1D1-huvBCD. Transcriptional start sites for each huvA, huvX, and tonB1 promoters were identified by primer extension analysis, and their respective -10 and -35 regions were shown to exhibit similarity to those of sigma70-recognized promoters. Expression from the three promoters, as analyzed by transcriptional fusions to a promoter less lacZ gene, was regulated by the iron concentration. Furthermore, analysis of the beta-galactosidase activities of these fusions in a V. anguillarum fur mutant demonstrated that the ferric uptake regulator repressor protein (Fur) is directly involved in the negative iron-mediated regulation of the heme uptake cluster.