Lactobacilli provide an important microbial defense against genital colonization by pathogens. The role of hydrogen peroxide (H2O2) in the control of genital microflora was explored in a cross-sectional study of 275 women in the second trimester of pregnancy. Vaginal cultures were obtained for detection of H2O2-positive and H2O2-negative lactobacilli and other members of the genital microflora. Compared with women with H2O2-negative lactobacilli, women colonized by H2O2-positive lactobacilli were less likely to have bacterial vaginosis, symptomatic candidiasis, and vaginal colonization byGardnerella vaginalis, Bacteroides, Peptostreptococcus, Mycoplasma hominis, Ureaplasma urealyticum,and viridans streptococci (P ≤ .05 for each comparison). In addition to the above organisms, women without vaginal lactobacilli were more likely than those women with H2O2-positive lactobacilli to haveChlamydia trachomatis,and less likely to be colonized byEnterococcusor coagulase-negative staphylococci (P < .05 for each comparison). Vaginal colonization by group B streptococci orEscherichia coliwas not related to the presence of H2O2-positive lactobacilli. These data suggest that the presence of H2O2-positive lactobacilli in the vagina is inversely correlated with infection by some genital pathogens in pregnant women.(Obstet Gynecol 1992;79:369-73)