Three-color flow cytometry immunophenotyping revealed significant increases of CD57+ and CD28− cells among both circulating CD4+ and CD8+ T lymphocytes of untreated hemato-oncological patients (n= 54) as compared to healthy donors (n= 55), with CD57 and CD28 expression on the patients’ T cells being largely reciprocal. Marked expansion of CD57+ cells among circulating CD4+ T lymphocytes was frequently detected in patients with chronic leukemia of B cell origin (B-CLL, hairy cell leukemia) but not in patients with chronic myeloid leukemia, suggesting a causal relation with the tumor’s major histocompatibility complex class II expression. Using immunomagnetic separation techniques, we further demonstrate that the patients’ CD57+/CD28− T cells display a typical Th1-type cytokine secretion profile upon anti-CD3 stimulation, with a markedly higher secretion of the Th1-type cytokines IL-2, IFN-γ, and TNF-α than their CD57−/CD28+ counterparts. Cytotoxic activity of circulating CD8+ T lymphocytes, measured ex vivo in an anti-CD3-redirected assay, was almost exclusively exerted by the CD57+/CD28− subset. Moreover, a marked cytotoxic activity was detected within CD4+ CD57+ T cells from some B-CLL patients. Finally, the patients’ CD57+/CD28− T cells displayed an increased tendency to apoptosis in culture. Collectively, our results indicate that the expanded CD57+/CD28− T cells in hemato-oncological patients represent differentiated effector cells, similar to their (quantitatively minor) counterpart in healthy donors. The reason for their expansion and their pathophysiologic significance, however, remains unclear.