<jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Vibrio anguillarum</jats:italic> , which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system ( <jats:italic>vanRI</jats:italic> ) and to produce <jats:italic>N</jats:italic> -(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a <jats:italic>vanI</jats:italic> null mutant still activated <jats:italic>N</jats:italic> -acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in <jats:italic>V. anguillarum</jats:italic> . In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as <jats:italic>N</jats:italic> -hexanoylhomoserine lactone (C6-HSL) and <jats:italic>N</jats:italic> -(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase <jats:italic>V. anguillarum</jats:italic> spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, <jats:italic>vanM</jats:italic> , the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the <jats:italic>luxL</jats:italic> and <jats:italic>luxM</jats:italic> genes, which are required for the production of <jats:italic>N</jats:italic> -(3-hydroxybutanoyl)homoserine lactone in <jats:italic>Vibrio harveyi</jats:italic> . However, resequencing of the <jats:italic>V. harveyi luxL/luxM</jats:italic> junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed <jats:italic>luxM</jats:italic> ). Downstream of <jats:italic>vanM</jats:italic> , we identified a homologue of <jats:italic>luxN</jats:italic> ( <jats:italic>vanN</jats:italic> ) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in <jats:italic>V. harveyi</jats:italic> . A mutation in <jats:italic>vanM</jats:italic> abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the <jats:italic>vanM</jats:italic> mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via <jats:italic>vanRI</jats:italic> . However, a <jats:italic>vanN</jats:italic> mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that <jats:italic>V. anguillarum</jats:italic> possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both <jats:italic>V. fischeri</jats:italic> (the LuxRI homologues VanRI) and <jats:italic>V. harveyi</jats:italic> (the LuxMN homologues, VanMN).