T-cells and natural killer (NK)-cells can be distinguished by their immunophenotype and molecular biological studies though there is overlap in T- and NK-cell antigen expression, function, and malignant diseases. The relatively new cell type of NKT-cells (also termed NK-like T-cells) represents a subpopulation of T-cells that share some characteristics with NK-cells. T- and NKT-cells have their T-cell receptor (TCR) genes rearranged while NK-cells are identified molecularly and immunologically by the absence of TCR gene rearrangements and TCR protein and lack of certain surface antigens. Various continuous malignant cell lines have been derived from patients with T-cell, NK- and NKT-cell neoplasms. These cell lines possess several traits typical of the respective diseases. Characterization of these cell lines which was the objective of this study will facilitate future studies of cell biology and therapeutics for which cell lines are indispensable models. In view of the imprecision of morphological criteria alone, we analyzed a series of seven NK-cell, five NKT-cell and five T-cell lines using functional and immunophenotypic tools. All T-cell lines were negative for the presence of azurophilic granules, NK activity and Epstein-Barr virus (EBV). In contrast, 7/7 NK-cell and 4/5 NKT-cell lines displayed the azurophilic granules but only three of these combined twelve NK/NKT-cell lines showed significant NK activity which may be explained by the functional immaturity of the cells. EBV was found in 5/7 NK-cell and in 1/5 NKT-cell lines. As expected, T-cell lines were commonly positive for T-cell surface antigens and negative for NK-cell markers, and NK-cell lines vice versa; nevertheless, a number of immunomarkers were shared between T- and NK-cell lines. NKT-cell lines express T-cell, NK-cell and markers shared between T- and NK-cells. Sets of markers distinctive for the three types of cell lines are presented. The composite data gained on the present panels of cell lines allow for the operational definition of typical NK- and NKT-cell line profiles. Such cell lines will prove invaluable as informative models for studies of normal and neoplastic NK- and NKT-cell biology.