Economic analyses of patients’ costs are pertinent to improve effective healthcare services including the prevention of mother-to-child HIV/AIDS transmission (PMTCT). This study assessed the direct and non-direct medical costs borne by pregnant women attending PMTCT services in urban (high-HIV prevalence) and rural (low-HIV prevalence) settings, in Ethiopia. Patient-level direct medical costs and direct non-medical data were collected from HIV-positive pregnant women in six regions. The cost estimation was classified as direct medical (service fee, drugs and laboratory) and direct non-medical (food, transportation and accommodation). The mean direct medical expense per patient per year was Ethiopian birr (ETB) 746 (US38)intheurbansettings,ascomparedtoETB368(US 19) in the rural settings. On average, a pregnant woman from urban and rural catchments incurred direct non-medical costs of ETB 6,435 (US327)andETB2,154(US 110) per year, respectively. On average, non-medical costs of friend/relative/guardian were ETB 2,595 (US132)andETB2,919(US 148.39) in the urban and rural settings, respectively. Although the PMTCT service is provided free of charge, HIV-positive pregnant women and infant pairs still face a substantial amount of out-of-pocket spending due to direct medical and non-medical costs.  
