Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.