The association of maternal and perinatal factors with mother-infant transmission of HIV-1 was examined in a prospective multicenter cohort of singleton live births to 508 HIV-1-infected women with children of known HIV-1 infection status (91 [18%] HIV-1-infected, 417 [82%] uninfected). From multivariate logistic regression, independent predictors of HIV-1 transmission included maternal CD4 percentage (CD4%) (odds ratio [OR] per 10% increase in CD4% = 0.70; p = .003), ruptured membranes <24 hours (OR = 3.15; p = .02), and maternal bleeding (OR = 2.90; p = .03), whereas maternal zidovudine (ZDV) use was marginally associated (OR = 0.60; p = .08). The associations of maternal urinary cytomegalovirus (CMV) shedding, oropharyngeal Epstein-Barr virus (EBV) shedding, and serology profiles during pregnancy with HIV-1 transmission were examined in the subset of mothers in whom the CMV and EBV measurements were available. Maternal EBV seropositivity, CMV shedding, and CMV seropositivity were 100% (279 of 279), 7% (16 of 229), and 92% (270 of 274), respectively. These rates did not differ between transmitting and nontransmitting mothers. In univariate analyses, maternal EBV shedding was higher among transmitting than nontransmitting mothers (40 of 49 [82%] compared with 154 of 226 [68%]; p = .06) and was independently associated with transmission in multivariate logistic analyses adjusting for CD4%, ruptured membranes, and ZDV use, with an OR of 2.45 (95% confidence interval (CI), 1.03-5.84; p = .04). This permits the conclusion that EBV shedding is associated with maternal-infant HIV-1 transmission, independent of CD4%.