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Background Limited information is available on anti-retroviral concentrations in women/infant pairs receiving prophylaxis for breastfeeding transmission of HIV and on the relationship between drug levels and the virological and haematochemistry parameters.Methods Patient population included HIV-positive pregnant women receiving antiretroviral prophylaxis from gestational week 25 until 6 months after delivery and their breastfed infants. Blood and breast milk samples were collected at delivery, and at months 1, 3 and 6 postpartum. Drug concentrations were measured by liquid chromatography–mass spectrometry.Results Overall, 66 women were studied: 29 received zidovudine (ZDV), lamivudine (3TC) and nevirapine (NVP), 28 stavudine (d4T), 3TC and NVP, and 9 ZDV, 3TC and lopinavir/ritonavir (LPV/r). Women who received >9 weeks of pre-partum prophylaxis were significantly more likely to have an undetectable viral load both in plasma and in breast milk at delivery. No emergence of resistance mutations was observed in breast milk. Breast milk/plasma concentration ratios were 0.6 for ZDV, 3TC and NVP, 1.0 for d4T and 0.4 for LPV/r. Only NVP reached significant levels in the infants. No correlation with any adverse events, including infant anaemia, was observed with drug concentrations. Two infants who acquired HIV infection had non-nucleoside reverse transcriptase inhibitor mutations at month 6.Conclusions Maternal administration of these three regimens up to 6 months postpartum was effective and safe for both mothers and infants. No significant correlation was found between drug concentrations and infant haematological parameters, supporting the hypothesis that other factors may contribute to the development of anaemia in these settings.