OBJECTIVES: To evaluate efficacy of the antenatal, intra-partum and post-natal antiretroviral components of a public service Prevention of Mother to Child (MTCT) program in infants. DESIGN: Analysis of prospectively collected screening data of demographic and MTCT-related interventions and HIV-infection status of infants identified through HIV-specific DNA polymerase chain reaction. SETTING: Tygerberg Children's Hospital, Western Cape Province, South Africa. SUBJECTS: HIV-infected women and their infants identified through participation in a public service MTCT program were referred for possible participation in a prospective study of isoniazid prophylaxis. INTERVENTIONS: Key components of the Program include voluntary counselling and testing, zidovudine to the mother from between 28 and 34 weeks gestation and to the newborn infant for the first week, single dose nevirapine to the mother in labour and the newborn shortly after birth and free formula for 6 months. MAIN OUTCOME MEASURES: Number and percentage of HIV-infected infants and extent of exposure to antenatal, intrapartum and post-natal antiretrovirals. RESULTS: Of 656 infants with a median age of 12.6 weeks, screened between April 1(st) 2005 through May 2006, 39 were HIV-infected giving a transmission rate of 5.9% (95% CI: 4.4% - 8.0%). Antenatal prophylaxis was significantly associated with reduced transmission (OR: 0.43 (95% CI: 0.21 - 0.94)) as opposed to intrapartum and postpartum components (p=0.85 and p=0.84, respectively). In multivariable analysis the antenatal component remained significant (OR=0.40 (95% CI 0.19 - 0.90)). CONCLUSIONS: The antenatal phase is the most important antiretroviral component of the MTCT program, allowing most opportunity for intervention.