Previous studies have shown that Gardnerella vaginalis can utilize human hemoglobin as a sole source of iron. In this study, the interaction between human hemoglobin and G. vaginalis cells was investigated. With a solid phase dot blot assay, G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled human hemoglobin. A human hemoglobin-binding protein with an estimated molecular weight of 124 kilodaltons (kDa) was detected by Western blot analysis of G. vaginalis proteins. The hemoglobin-binding activity of this protein was found to be heat stable and was observed in G. vaginalis cells grown under iron-restrictive and iron-replete conditions. The 124-kDa hemoglobin-binding protein was not detected from intact G. vaginalis cells treated with trypsin prior to Western blot analysis, suggesting that this protein was surface exposed.