A subset of peripheral blood T lymphocytes coexpressing CD3 and IgG Fc receptors (FcR) (CD16/Leu-11 antigen) have been identified, isolated, and functionally characterized. The CD3+, CD16+ cells were established in short-term culture using growth medium containing interleukin 2 (IL-2). Both the freshly isolated cells and the cultured cell line stably expressed the CD3+, CD16+ phenotype. Furthermore, a majority of these T cells lacked either CD4 or CD8 expression. Like in vitro-activated cytotoxic T lymphocytes and natural killer (NK) cells, the CD3+, CD16+ cells showed numerous azurophilic granules. Although these cells failed to mediate significant levels of NK cell-mediated cytotoxicity even after stimulation with IL-2, they efficiently functioned as effectors of antibody-dependent cellular cytotoxicity (ADCC). The Ig isotype specificity of the ADCC was analyzed using an isotype switch-variant family of a murine anti-HLA monoclonal antibody (mAb). Similar to the CD3-, CD16+ NK cell population, the CD3+, CD16+ T cells preferentially used the IgG2a antibody to mediate ADCC. The CD3+, CD16+ cells demonstrated a proliferative response when cocultured with either a NK-sensitive tumor cell line, K562, or a NK-insensitive B lymphoblastoid cell line, CCRF-SB. The response against CCRF-SB was significantly inhibited by anti-IL-2 receptor antibody, whereas the response against K562 was only partially diminished. Cytotoxicity was also induced in the CD3+, CD16+ population by the presence of anti-CD3 mAb, indicating that cytotoxicity can be triggered by stimulation via the CD3-T cell antigen receptor complex. By isolating these CD3+, CD16+ cells from the peripheral blood of a normal, healthy individual, it has been possible to extensively study the morphology, antigenic phenotype, and functional behavior of this unique subset of T lymphocytes expressing IgG FcR.