The application of bone marrow gene therapy has been stalled by the inability to achieve stable high-level gene transfer and expression in the totipotent stem cells. We show that retroviral vectors can stably introduce genes into antigen-specific murine and human T lymphocytes in culture. Murine helper T cells were transduced with the retroviral vector SAX to express both neomycin-resistance and human adenosine deaminase genes. These cells were expanded in culture and selected for expression of neomycin resistance with G418. The gene insertion, selection, and culture expansion did not alter antigen specificity or growth characteristics of the T cells in vitro. To determine if cultured T cells might be used for gene therapy, their persistence and continued expression of the introduced genes was evaluated in nude mice transplanted with the SAX-transduced T cells. G418-resistant cells could be readily recovered from the spleens of recipients of transduced T cells for several months. In addition, recovered cells continued to produce human adenosine deaminase. Based on these observations, we studied cultured human tumor-infiltrating lymphocytes as a candidate cell for a trial of gene transfer in man. Exponential cultures of interleukin-2-stimulated tumor-infiltrating lymphocytes were efficiently transduced with the neomycin-resistance gene using the retroviral vector N2. Gene insertion and subsequent G418 selection did not substantially alter the growth characteristics, interleukin 2 dependence, membrane phenotype, or cytotoxicity profile of the transduced T cells. These studies provided a portion of the experimental evidence supporting the feasibility of the presently ongoing clinical trials of lymphocyte gene therapy in cancer as well as in patients with adenosine deaminase deficiency.