To study the cause of nonspecific vaginitis, we analyzed vaginal fluid from normal women and from 53 women with nonspecific vaginitis, using quantitative anaerobic cultures and gas-liquid chromatography for short-chained organic-acid metabolites of the microbial flora. In normal vaginal fluid, lactate was the predominant acid, and the predominant organisms were lactobacillus and streptococcus species (lactate producers). In nonspecific vaginitis, lactate was decreased, whereas succinate, acetate, butyrate, and propionate were increased, the predominant flora included Gardnerella (Haemophilus) vaginalis (acetate producer), and anaerobes, which included bacteroides species (succinate producers) and peptococcus species (butyrate and acetate producers). After metronidazole therapy, symptoms and signs of nonspecific vaginitis cleared, butyrate and propionate disappeared, and lactate and lactate-producing organisms became predominant. We conclude that certain anaerobes act with G. vaginalis as causes of nonspecific vaginitis, and that a high ratio of succinate to lactate in vaginal fluid is a useful indicator in the diagnosis of this condition.