The clonogenic growth inhibition, the cell cycle dependence of N4-hexadecyl-1-beta-D-arabinofuranosylcytosine (NHAC) cytotoxicity and the capability to induce apoptosis in ara-C-sensitive and -resistant HL-60 cells were investigated and compared with arabinofuranosylcytosine (ara-C). In the clonogenic assay with sensitive HL-60 cells, ara-C was slightly more effective than a liposomal preparation of NHAC, whereas in the resistant cells, NHAC revealed its potency to overcome ara-C resistance, resulting in a 23-fold lower 50% inhibitory concentration compared with ara-C. Cell cycle dependent cytotoxicity and induction of apoptosis were studied by flow cytometry, using the bromodeoxyuridine-propidium iodide and terminal transferase method respectively. In contrast to ara-C, NHAC exerted no phase-specific toxicity at low concentrations (< 40 microM). At higher concentrations the S-phase-specific toxicity increased, probably resulting from ara-C formed from NHAC. NHAC induced apoptosis at higher drug concentrations than ara-C, however apoptosis appeared not to be limited to the S-phase cells. Apoptosis occurred in both cell lines within 2-4 h after drug exposure. These results give further evidence that NHAC exerts its cytotoxicity by different mechanisms of action than ara-C and might therefore be active in ara-C-resistant tumours.