Cell lines were established from purified large granular lymphocites (LGL) isolated from the peripheral blood of seven patients with phenotypically homogeneous LGL expansions. LGL were stimulated with phytohemagglutinin (PHA) or recombinant interleukin-2 (rIL-2) and further expanded in vitro in IL-2-containing media. The surface phenotype of LGL, as assessed by monoclonal antibody staining, was T3+ T8+ in five patients, T3- T8- in one, and T3+ T8- in another patient. The cells also expressed Leu 7, Leu 11, and/or OKM 1 markers in various proportions and were identifiable as LGL by their morphological and cytochemical features. The original surface phenotype of the unstimulated LGL was retained in the IL-2-dependent cell lines from each individual patient, i.e., T3+ T8+ cells originated T3+ T8+ cell lines and T3- T8- cells originated T3- T8- cell lines. Other markers, such as Leu 11 and OKM 1, were generally lost in culture. LGL proliferated in response to rIL-2 but did not express detectable IL-2 receptors, even after prolonged periods of culture. All cell lines from each individual patient had the same surface phenotype, and within the single lines, all of the cells expressed the same markers. Cell lines from two patients consistently displayed chromosomal abnormalities. Although different in the two patients, the abnormalities were identical in all of the lines from the same patient and detectable in most of the cells examined, suggesting a clonal origin for the abnormally expanded LGL populations. Freshly isolated LGL did not exert NK activity. However, the IL-2-dependent LGL lines acquired the ability to kill K562 target cells and to produce gamma interferon (γ-IFN). No direct correlation was observed between these two properties. © 1986 Plenum Publishing Corporation.