Vibrio anguillarum can utilize hemin and hemoglobin as sole iron sources. In previous work we identified HuvA, the V. anguillarum outer membrane heme receptor by complementation of a heme utilization mutant with a cosmid clone (pML1) isolated from a genomic library of V. anguillarum. In the present study, we describe a gene cluster contained in cosmid pML1, coding for nine potential heme uptake and utilization proteins: HuvA, the heme receptor; HuvZ and HuvX; TonB, ExbB, and ExbD; HuvB, the putative periplasmic binding protein; HuvC, the putative inner membrane permease; and HuvD, the putative ABC transporter ATPase. A V. anguillarum strain with an in-frame chromosomal deletion of the nine-gene cluster was impaired for growth with heme or hemoglobin as the sole iron source. Single-gene in-frame deletions were constructed, demonstrating that each of the huvAZBCD genes are essential for utilization of heme as an iron source in V. anguillarum, whereas huvX is not. When expressed in Escherichia coli hemA (strain EB53), a plasmid carrying the gene for the heme receptor, HuvA, was sufficient to allow the use of heme as the porphyrin source. For utilization of heme as an iron source in E. coli ent (strain 101ESD), the tonB exbBD and huvBCD genes were required in addition to huvA. The V. anguillarum heme uptake cluster shows some differences in gene arrangement when compared to homologous clusters described for other Vibrio species.