ObjectiveTo determine HIV-1 RNA load during the third trimester of pregnancy and evaluate its effect on in utero and intra-partum/postpartum transmissions in a breastfeeding population.DesignA nested case-control study within a PMTCT cohort of antiretroviral therapy naive pregnant women and their infants.MethodsA case was a mother who transmitted HIV-1 to her infant (transmitter) who was matched to one HIV-1 positive but non-transmitting mother (control).ResultsFrom a cohort of 691 pregnant women, 177 (25.6%) were HIV-1 positive at enrolment and from these 29 (23%) transmitted HIV-1 to their infants, 10 and 19 during in utero and intra-partum/postpartum respectively. Twenty-four mothers sero-converted after delivery and three transmitted HIV-1 to their infants. Each unit increase in log10 viral load was associated with a 178 cells/mm3 and 0.2 g/dL decrease in TLC and hemoglobin levels, p = 0.048 and 0.021 respectively, and a 29% increase in the risk of transmission, p = 0.023. Intra-partum/postpartum transmitters had significantly higher mean viral load relative to their matched controls, p = 0.034.ConclusionAntenatal serum HIV-1 RNA load, TLC and hemoglobin levels were significantly associated with vertical transmission but this association was independent of transmission time. This finding supports the rationale for preventive strategies designed to reduce vertical transmission by lowering maternal viral load.