The earliest, most reliable methods for detecting human immunodeficiency virus type 1 (HIV-1) infection in infants at risk of perinatal HIV-1 and risks for transmission were investigated. Fifteen infants were followed prospectively from birth to age 21 to 48 months. Epidemiologic data on mothers during pregnancy were documented, and maternal proviral load (by quantitative polymerase chain reaction) and viral phenotype by HIV isolation were performed. Infants were assessed clinically and HIV isolation, HIV p24 antigen, polymerase chain reaction and total serum immunoglobulin determinations were performed. Four infants were infected. HIV isolation, HIV p24 antigen and polymerase chain reaction were positive within 3 months in all infected infants (100% sensitivity). False positive rates for a single test were 18, 9 and 9%, respectively. Median age of clearance of maternal antibodies was 13.4 ± 2.1 months. Serum immunoglobulin G was significantly elevated after 6 months in all 4 infected infants. Advanced maternal age (≥30 years; Fisher's exact test,P> 0.014) was associated with transmission. A trend in higher maternal viral burden was observed among transmitters. Non-syncytium-inducing phenotype was present initially in all transmitting mothers.