Tumor immunotherapy has been limited to date by the poor antigenicity of most tumors, the immunocompromised state of many cancer patients, and the slow tumor penetration and short half-life of exogenously-introduced anti-tumor antibodies. Our group has developed a model immunotherapy system using a chimeric construct containing an antibody V region fused to a T cell activation molecule (T body) introduced by transfection into cytotoxic T cell lines, or populations of activated primary T or natural killer (NK) cells. In this study we have optimized the conditions needed for efficient transduction of human peripheral lymphocytes (PBL) using retroviral vectors pseudotyped with the gibbon ape leukemia virus (GaLV) envelope. Selection of packaging cells producing high virus titers was performed following transfection with constructs containing the green fluorescent protein (GFP), and FACS sorting. As a model chimeric receptor gene we used a tripartite construct consisting of a single-chain anti-TNP antibody variable region linked to part of the extracellular domain and the membrane spanning regions of the CD28 coreceptor molecule and joined at its 5' end to a gene fragment encoding the intracellular moiety of the gamma activation molecule common to the Fcepsilon and Fcgamma receptors. Enriched preparations of retrovectors containing this chimeric receptor and the GFP gene could stably and efficiently transduce human PBL co-activated by anti-CD3 and anti-CD28 antibodies. In routine experiments, the transgene was expressed in 35-70% of the human T cells. Such lymphocytes express the chimeric receptors on their surface and upon stimulation with hapten immobilized on plastic they can produce IL-2. Transfectomas activated in this manner also undergo specific proliferation in the absence of exogenous IL-2. Moreover, the transduced lymphocytes could effectively lyse target cells expressing the TNP hapten on their surface. These studies establish the conditions for the optimal transfection of effector lymphocytes to redirect them against a variety of tumor targets.