Iron concentration influences the expression of a number of genes involved in iron uptake and virulence in bacteria. In Escherichia coli, coordinate regulation of these genes by iron depends on the product of the fur gene, which acts as an iron-responsive, DNA-binding repressor protein. Several genes in Vibrio cholerae are also repressed by iron; and a fur gene, homologous to E. coli fur, has been previously cloned from this organism. The present study was undertaken to define the roles of Fur and iron in regulating gene expression in V. cholerae. V. cholerae strains with a mutation in fur by virtue of suicide plasmid integration into this gene showed derepressed expression of two previously characterized, iron-regulated genes, irgA and viuA, in high concentrations of iron; even in the fur mutants, however, residual two- to threefold regulation by iron persisted. The fur mutant strains constructed by suicide plasmid integration required antibiotic selection to maintain the mutation. To analyze further the effect of Fur and iron on gene regulation in V. cholerae without the need for antibiotic selection, we used in vivo marker exchange to construct a nonrevertible V. cholerae fur mutant. This V. cholerae fur mutant grew significantly less well in Luria-Bertani medium than the wild-type parent but grew slightly better than the wild type under iron-restricted conditions. The V. cholerae fur mutant was unable to utilize a number of carbon sources including glycerol, acetate, succinate, lactate, and fumarate, that supported growth of the wild-type strain on minimal media. We utilized two-dimensional gel electrophoresis of whole-cell protein extracts from the fur mutant and wild-type strains following growth in conditions of either low or high concentrations of iron to identify proteins regulated by iron and/or Fur. Twenty-two proteins were negatively regulated by iron in the wild-type strain but constitutively expressed in the fur mutant, consistent with the model of Fur as an iron-dependent repressor. However, many other proteins were regulated in a different manner by iron and/or Fur. Seventeen proteins were negatively regulated by iron but independent of Fur, suggesting the presence of an additional iron-dependent repressor(s). Six proteins were strongly iron regulated in the fur mutant but hardly expressed at all in the wild-type strain regardless of the iron concentration, suggesting an interaction between Fur and another iron regulatory mechanism. There were 11 proteins that were induced rather than repressed by iron, in four different regulatory classes. Gene regulation in V. cholerae by Fur and iron is much more complex than previously thought and is reminiscent of the Lrp regulon in E.coli.