Six Gardnerella vaginalis strains were examined for the ability to utilize various iron-containing compounds as iron sources. In a plate bioassay, all six strains acquired iron from ferrous chloride, ferric chloride, ferrous sulfate, ferric ammonium citrate, ferrous ammonium sulfate, bovine and equine hemin, bovine catalase, and equine, bovine, rabbit, and human hemoglobin. All six strains also acquired iron from human lactoferrin, but not from human transferrin, as determined by a liquid broth growth assay. Siderophore production was detected in eight G. vaginalis strains by the chrome azurol S universal chemical assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytoplasmic membrane proteins isolated from G. vaginalis 594 grown under iron-replete and iron-restricted conditions revealed several iron-regulated proteins ranging in molecular mass from 33 to 94 kDa. These results indicate that G. vaginalis may acquire iron from iron salts and host iron compounds.