Intra-amniotic administration of adeno-associated virus (AAV) vector may be an effective way to deliver gene therapy for treatment of congenital pulmonary and intestinal disorders. In an effort to understand potential barriers to intra-amniotic gene therapy better, we determined whether human amniotic fluid (AF) could act as an inhibitor of AAV2-mediated gene transfer. AF samples were obtained from 21 different human pregnancies during routine amniocentesis at 16-20 weeks of gestation. An immortalized fetal human tracheal epithelial cell line (FHTE) was infected with AAV2 containing a luciferase reporter gene driven by the SV40 promoter in the presence and absence of each AF sample. Inhibition of transgene expression was observed in 8 (38%) of the AF samples (inhibitory AF) and resulted in luciferase levels of only 1.4% +/- 0.6% of those obtained with infection in normal media. Infections in 13 samples (62%) resulted in transgene expression comparable or in excess of infection in media alone (noninhibitory AF). Removal of immunoglobulin G (IgG) from inhibitory AF samples with Protein A returned luciferase expression to control levels (119% +/- 37% of control), suggesting the possible presence of inhibiting antibody. Eleven of the AF samples were evaluated by enzyme-linked immunosorbent assay (ELISA) for specific anti-AAV antibodies. All noninhibitory AF samples were negative (titers of < 1:20; n = 3), and 6 of the 8 inhibitory samples contained specific anti-AAV antibodies at titers ranging from 1:40 to 1:160. These studies demonstrate that AF from some individuals contains AAV-specific IgG that can inhibit gene transfer.