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To evaluate uptake of HIV testing in a prevention of mother-to-child transmission program (PMTCT) in Lilongwe, Malawi from April 2002 until December 2006.Retrospective analysis of monthly reports from the beginning of the program.Four antenatal clinics in Lilongwe, Malawi.Pregnant women attending urban antenatal clinics in Lilongwe were invited to participate in a PMTCT program. Women were given information and education on antenatal care and PMTCT in groups of 8 to 12. Written informed consent for HIV testing was obtained privately. Women returned for the test result 1–2 weeks later. Mothers and infants were given the HIVNET 012 regimen. Rapid HIV testing and ‘opt-out’ testing were instituted in July 2003 and April 2005, respectively. Infants were tested using HIV DNA PCR and, if HIV positive, a CD4 cell percentage was obtained and the infants were referred for further medical evaluation and treatment.The program reached 20 000 pregnant women in the first 12 months. Acceptance of HIV testing increased from 45% to 73% (P < 0.001) when rapid, same day testing was instituted. When opt-out testing was instituted, 99% of the mothers agreed to testing. Of the infants tested, 15.5% were HIV positive.Rapid HIV testing using the opt-out method increased acceptance of HIV testing in the PMTCT program to 99% in urban Lilongwe, Malawi.