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To evaluate the effectiveness of maternal combination antiretroviral prophylaxis for prevention of mother-to-child transmission of HIV (PMTCT) in a program setting.Prospective cohort study.Nine primary care clinics in rural Zambia.Two hundred and eighty-four HIV-infected pregnant women at at least 28 weeks gestation initiating PMTCT services between April 2009 and January 2011 and their newborn infants.In four ‘intervention’ sites, PMTCT comprised universal combination antiretroviral prophylaxis (i.e. irrespective of CD4 cell count) from pregnancy until the cessation of breastfeeding. In five ‘control’ sites, women received antenatal zidovudine and peripartum nevirapine, the standard of care at the time. Prophylaxis during breastfeeding was not available in control sites.Cumulative infant HIV infection and death at 12 months postpartum.At 12 month postpartum, one of 104 (1.0%) infants born to mothers at the intervention sites were HIV-infected, compared with 14 of 116 (12.1%) receiving care in the control sites [relative risk (RR): 12.6, 95% CI: 2.2–73.1; P = 0.005]. When we considered the composite outcome of HIV infection or death, similar trends were observed in the overall study population (RR: 3.4, 95% CI: 1.6–7.6; P = 0.002) and in a sub-analysis of women with CD4 cell count more than 350 cells/μl (RR: 3.2; 95% CI: 1.1–9.6; P = 0.04).When compared with PMTCT services based on antenatal zidovudine and peripartum nevirapine, the provision of maternal combination prophylaxis imparted measurable health benefits to HIV-exposed infants. Implementation research is needed to further tailor and optimize these strategies for similar field settings.