BACKGROUND: The human immunodeficiency virus (HIV) continues to be a major cause of maternal and infant mortality and morbidity in sub-Saharan Africa. Prevention of mother-to-child transmission of HIV (PMTCT) strategies have proven effective in decreasing the number of children infected in utero, intrapartum and during the breastfeeding period. This qualitative study explores knowledge and perceptions of HIV amongst pregnant women, healthcare workers' experiences of the national PMTCT services, and barriers to PMTCT, during a period of programme scale-up in urban Guinea-Bissau (2010-11). METHODS: In-depth interviews were undertaken amongst 27 women and 19 key informants at local antenatal clinics and the national maternity ward in Bissau, Guinea-Bissau. RESULTS: Amongst women who had been tested for HIV, awareness and knowledge of HIV and PMTCT remained low. Testing without informed consent was reported in some cases, in particular when the test was performed around the time of delivery. Possible drivers of inadequate counselling included lack of confidentiality, suboptimal healthcare worker training, lack of time, and perceived occupational risk. Demand-side barriers to PMTCT included lack of HIV and PMTCT knowledge, customary and cultural beliefs associated with HIV and ill-health, HIV stigma and discrimination, and fear of partnership dissolution. CONCLUSIONS: Socio-cultural and operational challenges, including HIV testing without informed consent, present significant barriers to the scale-up of PMTCT services in Bissau. Strengthening local capacity for effective counselling and testing in the antenatal setting is paramount. Further research into local customary beliefs relating to HIV is warranted.