<jats:title>ABSTRACT</jats:title> <jats:p> In response to low iron availability, <jats:italic>Vibrio parahaemolyticus</jats:italic> synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, <jats:sc>l</jats:sc> -alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the <jats:italic>pvuA</jats:italic> gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the <jats:italic>pvuA</jats:italic> gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named <jats:italic>pvsABCDE</jats:italic> , constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes <jats:italic>pvsABDE</jats:italic> are implicated in the biosynthesis of the siderophore. Another gene in the same operon, <jats:italic>pvsC</jats:italic> , encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named <jats:italic>pvuBCDE</jats:italic> , encode proteins strongly homologous to <jats:italic>Escherichia coli</jats:italic> FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, <jats:italic>psuA</jats:italic> and <jats:italic>pvuA</jats:italic> . Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.