We examined the effectiveness of various anti-tumour agents to natural killer (NK)-cell tumour cell lines and samples, which are generally resistant to chemotherapy, using flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay. Although NK-YS and NK-92 were highly resistant to various anti-tumour agents, l-asparaginase induced apoptosis in these two NK-cell lines. NK-cell leukaemia/lymphoma and acute lymphoblastic leukaemia (ALL) samples were selectively sensitive to l-asparaginase and to doxorubicin (DXR) respectively. Samples of chronic NK lymphocytosis, an NK-cell disorder with an indolent clinical course, were resistant to both drugs. Our study clearly separated two major categories of NK-cell disorders and ALL according to the sensitivity to DXR and l-asparaginase. We examined asparagine synthetase levels by real-time quantitative polymerase chain reaction (RQ-PCR) and immunostaining in these samples. At least in nasal-type NK-cell lymphoma, there was a good correlation among asparagine synthetase expression, in vitro sensitivity and clinical response to l-asparaginase. In aggressive NK-cell leukaemia, although asparagine synthetase expression was high at both mRNA and protein levels, l-asparaginase induced considerable apoptosis. Furthermore, samples of each disease entity occupied a distinct area in two-dimensional plotting with asparagine synthetase mRNA level (RQ-PCR) and in vitrol-asparaginase sensitivity (TUNEL assay). We confirmed rather specific anti-tumour activity of l-asparaginase against NK-cell tumours in vitro, which provides an experimental background to the clinical use of l-asparaginase for NK-cell tumours.