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To assess differences between HIV prevalence estimates derived from national population surveys and antenatal care (ANC) surveillance sites and to improve the calibration of ANC-derived estimates in Spectrum 2013 to more appropriately account for differences between these data.Retrospective analysis of national population survey and ANC surveillance data from 25 countries with generalized epidemics in sub-Saharan Africa and 8 countries with concentrated epidemics.Adult national population survey and ANC surveillance HIV prevalence estimates were compared for all available national population survey data points for the years 1999–2012. For sub-Saharan Africa, a mixed-effects linear regression model determined whether the relationship between national population and ANC estimates was constant across surveys. A new calibration method was developed to incorporate national population survey data directly into the likelihood for HIV prevalence in countries with generalized epidemics. Results were used to develop default rules for adjusting ANC data for countries with no national population surveys.ANC surveillance data typically overestimate population prevalence, although a wide variation, particularly in rural areas, is observed across countries and survey years. The new calibration method yields similar point estimates to previous approaches, but leads to an average 44% increase in the width of 95% uncertainty intervals.Important biases remain in ANC surveillance data for HIV prevalence. The new approach to model-fitting in Spectrum 2013 more appropriately accounts for this bias when producing national estimates in countries with generalized epidemics. In countries with concentrated epidemics, local sex ratios should be used to calibrate ANC surveillance estimates.