Purpose: The objective of this study was to examine lamivudine (3TC), zidovudine (ZDV), nelfinavir (NFV), and its active nelfinavir metabolite (M8) concentrations in paired maternal plasma and amniotic fluid samples to determine antiretroviral penetration or accumulation in the fetal compartment. Method: Ten paired amniotic fluid and maternal plasma samples were obtained during caesarian section for pharmacokinetic analysis. Antiretroviral concentrations were measured in both matrices using high-performance liquid chromatography (HPLC) and mass spectrometry (LC/MS) methodologies. Results: Median maternal plasma concentrations for NFV, M8, 3TC, and ZDV were 456, 244, 176, and 794 ng/mL, respectively, while median amniotic fluid concentrations were 118, 21, 2537, and 1483 ng/mL, respectively. The median NFV amniotic fluid to maternal plasma ratio was 0.44; the median M8 ratio was 0.11. Median 3TC and ZDV amniotic fluid to plasma ratios were 11.9 and 1.5, respectively. Conclusions: NFV and M8 exhibited partial drug transfer and/or accumulation in the amniotic compartment, whereas ZDV and 3TC concentrations mostly exceeded that in maternal plasma. Overall, all drugs achieved exposures in the amniotic fluid in excess of their wild-type viral susceptibilities. Amniotic fluid is an important compartment in the prevention of mother-to-child transmission; a further understanding of protease inhibitor and other antiretroviral drug penetration into amniotic fluid is warranted. © 2009 Thomas Land Publishers, Inc.