This study was conducted to identify reasons for a high and progressive loss to follow-up among HIV-positive mothers within a prevention-of-mother-to-child HIV transmission (PMTCT) program in a rural district hospital in Malawi. Three focus group discussions were conducted among a total of 25 antenatal and post-natal mothers as well as nurse midwives (median age 39 years, range 22-55 years). The main reasons for loss to follow-up included: (1) not being prepared for HIV testing and its implications before the antenatal clinic (ANC) visit; (2) fear of stigma, discrimination, household conflict and even divorce on disclosure of HIV status; (3) lack of support from husbands who do not want to undergo HIV testing; (4) the feeling that one is obliged to rely on artificial feeding, which is associated with social and cultural taboos; (5) long waiting times at the ANC; and (6) inability to afford transport costs related to the long distances to the hospital. This study reveals a number of community- and provider-related operational and cultural barriers hindering the overall acceptability of PMTCT that need to be addressed urgently. Mothers attending antenatal services need to be better informed and supported, at both community and health-provider level.