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Pregnant women have a significantly higher risk of HIV acquisition during gestation than their non-pregnant counterparts due to behavioral and biological factors. Acute seroconversion during gestation results in increased HIV mother-to-child transmission rates and has been identified as a major public health challenge. In order to address potential HIV seroconversion in our pregnant patients, we conducted a prospective cohort study to evaluate the acceptability of offering HIV testing to sexual partners of HIV-negative pregnant women receiving antenatal care at two hospitals in Porto Alegre, Brazil. Over a 14-month study period, HIV-negative pregnant women at two hospital-based clinic sites were encouraged to bring their stable sexual partner for HIV voluntary counseling and testing during prenatal care. Women were re-interviewed following delivery to measure success of the intervention. Of the 1223 HIV-negative pregnant women enrolled in the study, 663 (54%) of their male sexual partners received HIV testing during antenatal care and 4 (0.6%) were diagnosed with HIV infection. A total of 645 women were interviewed at the time of delivery, with 620 (97%) confirming that HIV testing was suggested to their partner. The most common reason provided by women as to why partners did not come for testing was work (69%) and lack of perceived risk (14%). Independent predictors of successful partner testing included being white (odds ratio [OR] 1.59, 95% confidence interval [CI] 1.18-2.12), married (OR 1.78, 95% CI 1.08-2.94), having an older age of sexual debut (OR 0.94, 95% CI 0.9-0.98), and being recruited at Hospital Conceiçao (OR 2.1, 95% CI 1.52-2.88). We conclude that HIV partner testing during prenatal care is acceptable, rendering this intervention attractive to public health programs targeting prevention of sexually transmitted infections.