To determine the association between genital tract shedding of human immunodeficiency virus (HIV) type 1 and vertical transmission, a case-control substudy was conducted within the Women and Infants Transmission Study. Antenatal cervicovaginal lavage specimens were assessed for HIV-1 RNA in the supernatant and HIV-1 RNA and DNA in cell pellets. Multivariate analyses compared 26 women who transmitted HIV to their infants with 52 women who did not; 33% received combination antiretroviral therapy, and 65% received monotherapy. Adjusted odds ratios (ORs) for the presence (OR, 3.42; 95% confidence interval [CI], 0.76-15.4; P=.11) and titer (OR, 1.66; 95% CI, 0.93-2.99; P=.09) of HIV-1 DNA suggested that there is an independent association with vertical transmission. When analyses were restricted to vaginal and nonelective cesarean deliveries, each one-log increase in mean titer of HIV-1 DNA was associated with a significantly higher risk of transmission (OR, 2.28; 95% CI, 1.09-4.78; P=.03). The cell-associated genital tract compartment is important in the pathophysiology and prevention of vertical HIV-1 transmission.