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Natural killer (NK) cells are a distinct lymphocyte subset that, like cytotoxic T cells, recognize and destroy abnormal self-cells. Unlike T cells, however, NK cells lack a uniquely rearranged antigen receptor gene or a single defining immunophenotypic attribute. Furthermore, there is considerable functional and phenotypic overlap between NK cells and cytotoxic T cells, and the latter are induced by cellular activation to express many NK-associated antigens. These factors all contribute to difficulties in recognizing abnormal NK-cell expansions and distinguishing them from cytotoxic T cells in the clinical laboratory. Recently, new classes of NK-associated major histocompatibility complex receptors have been described that can be of value in evaluating NK-cell and cytotoxic T-cell populations. In this review, immunophenotyping approaches using antibodies to traditional and novel T- and NK-associated antigens that can be used to evaluate these cell types and diagnose NK-cell and cytotoxic T-cell disorders in the clinical laboratory setting are discussed.