CONTEXT: Single-dose intrapartum and neonatal nevirapine (NVP) reduces perinatal HIV transmission and is in increasingly common use throughout the developing world. OBJECTIVE: We studied risk factors for perinatal transmission in the setting of NVP. DESIGN AND SETTING: A prospective cohort study at two public obstetrical clinics in Lusaka, Zambia. PATIENTS AND METHODS: In a volunteer sample of HIV-infected pregnant women and their newborns, the women received a 200 mg oral dose of NVP at the onset of labor; their infants received 2 mg/kg of NVP syrup within 24 h of birth. The main outcome measure was the infant HIV infection status at 6 weeks of life, determined by DNA polymerase chain reaction. RESULTS: Only 31 of 278 (11.2%) infants were infected at 6 weeks. In logistic regression, viral load exceeding the median [adjusted odds ratio (AOR), 3.1; 95% confidence interval (CI), 1.1-8.7] and 1 h or less elapsing between NVP ingestion and delivery (AOR, 5.0; 95% CI, 1.8-14) were associated with transmission. Women delivering within 1 h of NVP ingestion had a lower mean drug concentration (351 versus 942 ng/ml; P<0.001) and were more likely to have a 'sub-therapeutic' NVP level of less than 100 ng/ml (56 versus 20%; P<0.001) than those who delivered more than 1 h post-ingestion. However, concentrations <100 ng/ml were not more likely to be associated with transmission than concentrations > or = 100 ng/ml (12.9 versus 11.7%; P=0.8). We did not identify a threshold concentration below which risk of transmission increased. CONCLUSIONS: We confirmed low perinatal transmission rates with single-dose NVP. At least 1 h of pre-delivery NVP prophylaxis was a critical threshold for efficacy.