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 by Gardnerella vaginalis, Bacteroides, Peptostreptococcus, Mycoplasma hominis, Ureaplasma urealyticum, and viridans streptococci (P less than or equal to .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 have Chlamydia trachomatis, and less likely to be colonized by Enterococcus or coagulase-negative staphylococci (P less than .05 for each comparison). Vaginal colonization by group B streptococci or Escherichia coli was 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.