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Transmission through breast-feeding is an important cause of infant HIV-1 infections in developing countries; however, its mechanism remains largely unknown. We have explored the association between cell-free virus (CFV) and cell-associated virus (CAV) levels in breast milk (BM), as reflected by viral RNA and proviral DNA, respectively, and the risk of infant HIV-1 infection after 6 weeks postpartum.Sixty-one HIV-positive mothers who transmitted HIV-1 by BM were matched to 61 HIV-positive nontransmitting mothers based on their infant's age at sample collection. CFV and CAV were quantified in a single milk specimen per mother preceding the infant's first HIV-positive result.After adjusting for maternal CD4+ cell counts and disease stage, each 10-fold increase in CFV or CAV load was associated with an almost 3-fold increase in BM transmission. Whereas CAV load was predictive of transmission before and after 9 months postpartum, CFV was a significant predictor of transmission occurring only after 9 months. Phylogenetic analyses of the C2 to C5 env region showed that 85% of infants (11 of 13 infants) harboring viruses that clustered with CFV in their mother's milk were infected after 9 months postpartum.A reduction in milk CAV and CFV loads might significantly decrease HIV-1 transmission by breast-feeding.