Vectors based on human adenovirus (Ad) and adeno-associated virus (AAV) are being evaluated for human gene therapy. The response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. We have surveyed cohorts of normal subjects and cystic fibrosis patients for pre-existing immunity to these viruses, caused by naturally acquired infections. A number of humoral and cellular assays to adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 2 (AAV2) were performed from serum and peripheral blood mononuclear cells. Virtually all subjects had Ig to Ad5 although only 55% of these antibodies neutralized virus (NAB). Approximately two of three patients demonstrated CD4+ T cells that proliferated to Ad antigens of which most were of the TH1 subset, based on cytokine secretion. A substantially different pattern of immune responses was observed to AAV2. Although virtually all patients had Ig to AAV2, most of these antibodies were not neutralizing (32% NAB) and only 5% of patients had peripheral blood lymphocytes that proliferated in response to AAV2 antigens. These studies demonstrate marked heterogeneity in pre-existing immunity to Ad5 and AAV2 in human populations. The impact of these findings on outcome following gene therapy will require further study.