Recent studies suggest that acquisition of HIV-1 infection during pregnancy and breastfeeding is associated with a high risk of HIV mother-to-child transmission. This study evaluates risk factors associated with HIV acquisition during pregnancy in women delivering at a large metropolitan medical facility located in the south of Brazil. From February to August 2009, our group conducted a cross-sectional study assessing women's risk for HIV acquisition by administering an oral survey to peripartum women. Of 2465 participants, 42% (n = 1046) knew that partner had been tested for HIV. During pregnancy, 82% (n = 2022) of participants never used condoms; yet 97% (n = 2399) practiced vaginal sex. Multivariate logistic regression analysis showed that patients with more years of education, in a relationship for more than 1 year, and who knew their own HIV status were more likely to know their partners' HIV status (P < 0.05). Those who were in relationship for more than 1 year and were married/living together were more likely to be comfortable discussing HIV testing with partners (P < 0.05). In conclusion, women in Brazil are at risk of HIV-infection during pregnancy as they remain sexually active, often do not know their sexual partner's HIV status, and have minimal condom use.