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α-Defensins have been observed to have anti-HIV activity but have not been investigated in relation to mother-to-child HIV transmission. We measured the concentration of α-defensins in breast milk of HIV-positive mothers and tested whether the concentrations were associated with HIV transmission. A nested case-control study of 32 HIV-positive women who transmitted HIV to their infants and 52 randomly selected HIV-positive women who did not transmit HIV to their infants was conducted in Lusaka, Zambia. α-Defensins were detected in most (79%) of the milk samples tested. Concentrations of α-defensins increased as breast milk HIV RNA quantity increased, and breast milk HIV RNA quantity was, in turn, a strong and significant predictor of HIV transmission. After adjustment for milk HIV RNA quantity, however, α-defensin concentration was significantly associated with a decreased risk of intrapartum and postnatal HIV transmission (odds ratio = 0.3, 95% confidence interval: 0.09-0.93). Our data suggest that there may be a role for α-defensins in prevention of HIV transmission to breastfed infants.