Context: Understanding the risk of human immunodeficiency virus (HIV) transmission through breastfeeding is essential for advising HIV-infected mothers and formulating public health policy recommendations. Objective: To measure the frequency, timing, and risk factors of HIV transmission through breast milk. Design: Prospective cohort study conducted between 1994 and 1997, with follow-up of infants through 24 months of age. Setting: Postnatal clinic of tertiary care hospital, Blantyre, Malawi. Participants: A total of 672 infants (HIV-negative at birth) born to HIV-infected women who had not received antiretroviral drugs during or after pregnancy. Main Outcome Measure: Incidence of HIV in breastfed infants by age and maternal and infant risk factors for HIV transmission, using proportional hazard models to derive risk ratios (RRs) and 95% confidence intervals (CIs). Results: Forty-seven children became HIV-infected while breastfeeding but none after breastfeeding had stopped. The cumulative infection rate while breastfeeding, from month 1 to the end of months 5, 11, 17, and 23, was 3.5%, 7.0%, 8.9%, and 10.3%, respectively. Incidence per month was 0.7% during age 1 to 5 months, 0.6% during age 6 to 11 months, and 0.3% during age 12 to 17 months (P= .01 for trend). The only factors significantly associated with low risk of postnatal HIV transmission in a multivariate model were high maternal parity (RR, 0.23; 95% CI, 0.09-0.56) and older maternal age (RR, 0.44; 95% CI, 0.23-0.84). Conclusions: Our data suggest that the risk of HIV infection is highest in the early months of breastfeeding, which should be considered in formulating breastfeeding policy recommendations.