The prevention of mother-to-child transmission of HIV-1 during breastfeeding is a major concern in resource-poor settings where alternatives to breast milk may be unaffordable, unsafe and limited by social stigma. The use of triple-drug antiretroviral regimens initiated during pregnancy and continued throughout breastfeeding is being studied as a means to prevent transmission by reducing HIV-1 viral load in the maternal serum and breast milk. Studies characterizing the exposure of antiretroviral agents in breast milk and in the breastfed infant are important to understand the dynamics of HIV-1 replication in breast milk and to establish the safety profiles of antiretroviral drugs.