Lactobacillus acidophilus has been reported to be the predominant vaginal species. Vaginal lactobacilli isolated from 215 sexually active women were identified using whole-chromosomal DNA probes to 20 American Type Culture Collection Lactobacillus strains. Most women were colonized by L. crispatus (32%), followed by L. jensenii (23%), a previously undescribed species designated L. 1086V (15%), L. gasseri (5%), L. fermentum (0.3%), L. oris (0.3%), L. reuteri (0.3%), L. ruminis (0.3%), and L. vaginalis (0.3%). H2O2 was produced by 95% of L. crispatus and 94% of L. jensenii isolates, compared with only 9% of L. 1086V. Colonization by L. crispatus or L. jensenii was positively associated with being white (P<.001), age >/=20 years (P=.05), barrier contraceptive usage (P=.008), and lower frequency of bacterial vaginosis (P<.001) and gonorrhea (P=.03). L. crispatus and L. jensenii, not L. acidophilus, are the most common species of vaginal lactobacilli.