Patients with T gamma lymphocytosis are a heterogeneous group, clearly distinguishable from other patients with chronic T lymphoproliferative disorders and usually without proven malignancy. We have attempted in vitro cloning of lymphocytes from three patients with an expansion of phenotypically and functionally different types of T gamma cells. One had T3+ B73.1+ T4 T8+ OKM1+ T gamma cells exerting antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell cytotoxicity; another had T3+B73.1-T4-T8+OKM1-ADCC+NK- and a third had T3-B73.1+T4-T8- OKM1+ADCC+NK+ cells. On morphological characterization, most of the mononuclear cells of these patients resembled large granular lymphocytes (LGLs). Although lymphocytes of these patients showed almost no proliferative response capacity after stimulation with mitogens, they shared the capacity to proliferate after stimulation with Epstein-Barr virus-transformed lymphoblastoid B (B-LCL) feeder cells. Stable clones were established by this procedure. Clones from patient 3 exerted cytolytic activity against a broad spectrum of tumor cell lines, including fresh biopsy specimens of melanoma tumor target cells. All of these clones (termed activated killer [AK] cells) had the surface phenotype T3-, T4-, T8- or +, HNK1-, OKM1-, Lyt3+, WT1+ and showed ADCC in addition to AK cell cytotoxicity. Most of them were B73.1+ and expressed IgG-Fc receptors. They most likely belong to the T cell lineage, since they express IL2 receptors as recognized by the Tac antibody and did not bind monoclonal antibodies directed against monocytes or granulocytes. Thus lymphocytes with the functional and phenotypical characteristics of T gamma cells can be cloned and expanded in vitro from the peripheral lymphocytes of these patients by using the appropriate stimulus. Our results indicate that, of the heterogeneous population of NK cells, the T3- cells are more rapidly expanded than T3+ subsets. It is discussed whether or not our culture system might selectively induce proliferation in “normal” T cells rather than aberrant ones.