The objective of this study was to investigate for the first time tenofovir (TFV) pharmacokinetics in plasma and peripheral blood mononuclear cells (PBMCs) of the neonate. HIV-1-infected pregnant women received two tablets of tenofovir disoproxil fumarate (TDF; 300 mg) and emtricitabine (FTC; 200 mg) at onset of labor and then one tablet daily for 7 days postpartum. A single dose of 13 mg/kg of body weight of TDF was administered to 36 neonates within 12 h of life after the HIV-1-infected mothers had been administered two tablets of TDF-emtricitabine at delivery. A total of 626 samples collected within the 2 days after the drug administration were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and analyzed by a population approach. In the neonate, the median TFV plasma area under the curve and minimal and maximal concentrations, respectively, were 3.73 mg/liter · h and 0.076 and 0.29 mg/liter. In PBMCs, TFV concentrations were detectable in all fetuses, whereas tenofovir diphosphate (TFV-DP) was quantifiable in only two fetuses, suggesting a lag in appearance of TFV-DP. The median TFV-DP neonatal concentration was 146 fmol/10⁶ cells (interquartile range [IQR], 53 to 430 fmol/10⁶ cells); two neonates had very high TFV-DP concentrations (1,530 and 2963 fmol/10⁶ cells). The 13-mg/kg TDF dose given to neonates produced plasma TFV and intracellular active TFV-DP concentrations similar to those in adults. This dose should be given immediately after birth to reduce the delay before the active compound TFV-DP appears in cells.