Background: Despite the availability of effective interventions for the prevention of mother-to-child transmission (PMTCT), questions remain regarding implementation of programmes in settings with limited resources. This article sets out to describe the first 2 years of the implementation of the national PMTCT programme in Ukraine. Methods: National data sources and data from a cohort of pregnant HIV-infected women delivering in 13 centres in Ukraine since 2000 were analysed. Results: Interventions for prevention of MTCT have been implemented as a national programme within Ukraine's well developed infrastructure for maternal and child health. Implementation of an ‘opt-out’ model of counselling and HIV testing in antenatal clinics resulted in a 97% uptake of women who agreed to be HIV tested. In 2002, ~91% of HIV-positive pregnant women received ARV prophylaxis (mainly single-dose nevirapine or short-course zidovudine) for PMTCT. The MTCT rate has decreased from 30% in 2000 to 10% in 2002. The need to scale-up prevention interventions in pregnant women with risky behaviour and late access to medical services was identified in a review of the national programme in 2003. Conclusions: Further implementation of a comprehensive approach for the prevention of HIV infection in infants, including more extensive ART regimen, as recommended by WHO, would help Ukraine to achieve the strategic goal of virtual elimination of HIV infection in infants by 2010.