HIV acquisition is significantly higher during pregnancy than in the postpartum period. We did a prospective study to estimate HIV incidence rates during pregnancy and lactation. Methods: We assessed 2188 HIV- negative sexually active women with 2625 exposure intervals during pregnancy and 2887 intervals during breastfeeding, and 8473 non-pregnant and non-lactating women with 24 258 exposure intervals. Outcomes were HIV incidence rates per 100 person years and incidence rate ratios estimated by Poisson multivariate regression, with the non-pregnant or non-lactating women as the reference group. We also assessed the husbands of the married women to study male risk behaviours. Findings: HIV incidence rates were 2.3 per 100 person years during pregnancy, 1.3 per 100 person years during breastfeeding, and 1.1 per 100 person years in the non-pregnant and non-lactating women. The adjusted incidence rate ratios were 2.16 (95% CI 1.39- 3.37) during pregnancy and 1.16 (0.82- 1.63) during breastfeeding. Pregnant women and their male partners reported significantly fewer external sexual partners than the other groups. In married pregnant women who had a sexual relationship with their male spouses, the HIV incidence rate ratio was 1.36 (0.63- 2.93). In married pregnant women in HIV- discordant relationships (ie, with HIV- positive men) the incidence rate ratio was 1.76 (0.62- 4.03). Interpretation: The risk of HIV acquisition rises during pregnancy. This change is unlikely to be due to sexual risk behaviours, but might be attributable to hormonal changes affecting the genital tract mucosa or immune responses. HIV prevention efforts are needed during pregnancy to protect mothers and their infants. We did a prospective study to estimate HIV incidence rates during pregnancy and lactation. Methods: We assessed 2188 HIV-negative sexually active women with 2625 exposure intervals during pregnancy and 2887 doses during breastfeeding , and 8473 non-pregnant and non-lactating women with 24 258 exposure intervals. Outcomes were HIV incidence rates per 100 person years and incidence rate ratios estimated by Poisson multivariate regression, with the non-pregnant or non-lactating women as the reference group Findings: HIV incidence rates were 2.3 per 100 person years during pregnancy, 1.3 per 100 person years during breastfeeding, and 1.1 per 100 person years in the non-pregnant and The adjusted incidence rate rates were 2.16 (95% CI 1.39-3.37) during pregnancy and 1.16 (0.82-1.63) during breast Pregnant women and their male partners reported significantly fewer external sexual partners than the other groups. In married pregnant women who had a sexual relationship with their male spouses, the HIV incidence rate was 1.36 (0.63- 2.93). In married pregnant women in HIV- discordant relationships (ie, with HIV-positive men) the incidence rate ratio was 1.76 (0.62- 4.03). Interpretation: The risk of HIV acquisition rises during pregnancy. This change is unlikely to be due to sexual risk behaviours, but might be attributable to hormonal changes affecting the genital tract mucosa or immune responses. HIV prevention efforts are needed during pregnancy to protect mothers and their infants.