Bacterial vaginosis is characterized microbiologically by replacement of the Lactobacillus-predominant vaginal flora by Gardnerella vaginalis, Bacteroides species, Mobiluncus species, and genital mycoplasmas. A standardized, laboratory-based diagnostic test for bacterial vaginosis is desirable in those instances in which a microscope is unavailable in the clinic or when the evaluator of the wet mount examination is inexperienced at recognizing clue cells. Vaginal cultures have excellent sensitivity for the diagnosis of bacterial vaginosis, but because the predictive value of a positive G. vaginalis culture is less than 50%, cultures are not recommended. Vaginal Gram smears are objective and reproducible, have 62% to 100% sensitivity, and have a positive predictive value of 76% to 100%. Diagnosis of bacterial vaginosis with use of Papanicolaou-stained smears has been reported, but standardized criteria have not been widely adopted. A rapid, office-based oligonucleotide probe test for high concentrations of G. vaginalis has been developed and may be useful when direct microscopy is unavailable or confidence in microscopic examination is low. Laboratory tests that detect microbial products unique to the vaginal fluid of women with bacterial vaginosis include detection of amines (putrescine, cadaverine, and trimethylamine), measurement of the relative levels of succinate and lactate in the vaginal fluid by gas chromatography, detection of proline aminopeptidase by colorimetric assay, or detection of sialidases in the vaginal fluid. Although these tests are not yet applicable to routine use, these research tests could be adapted for wider use in office laboratories. Vaginal Gram stains are more useful than culture for laboratory confirmation of bacterial vaginosis.