Immune responses to the therapeutic gene product are a potentially serious complication in treatment of genetic disease by gene therapy. Induction and maintenance of immunologic hypo-responsiveness to the therapeutic antigen is therefore critical to the success of gene-based treatment of inherited protein deficiency. Here, we demonstrate induction of antigen-specific CD4⁺ T-cell tolerance to a secreted transgene product (ovalbumin, ova) in ova-specific T-cell receptor (TCR) transgenic mice by hepatic adeno-associated virus (AAV)–mediated gene transfer. Transduced mice maintained stable circulating ova levels without evidence of an immune response. Lymph node cells and splenocytes were hypo-responsive to ova as early as day 10 after gene transfer. Numbers of TCR⁺CD4⁺ cells were reduced in secondary lymphoid organs and in the thymus by 1 to 2 months after vector administration. The remaining TCR⁺CD4⁺ cell population was anergic to ova antigen in vitro and enriched for CD25⁺ cells. These data provide direct evidence that transgene expression following in vivo viral gene transfer can induce CD4⁺ T-cell tolerance to the transgene product, involving anergy and deletion mechanisms.