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A problem commonly encountered in programs for prevention of mother-to-child-transmission (PMTCT) of HIV in sub-Saharan Africa is low rates of HIV test acceptance among pregnant women. In this study, we examined risk factors and reasons for HIV test refusal among 432 women attending three antenatal care clinics offering PMTCT in urban and semi-urban parts of the Mbarara district, Uganda. Structured interviews were performed following pre-test counselling. Three-hundred-eighty women were included in the study, 323 (85%) of whom accepted HIV testing. In multivariate analysis, testing site (Site A: OR = 1.0; Site B: OR = 3.08; 95%CI: 1.12–8.46; Site C: OR = 5.93; 95%CI: 2.94–11.98), age between 30 and 34 years (<20 years: OR = 1.0; 20–24 years: OR = 1.81; 95%CI: 0.58–5.67; 25–29 years: OR = 2.15; 95%CI: 0.66–6.97; 30–34 years: OR = 3.88; 95%CI: 1.21–13.41), mistrust in reliability of the …