Differentiation of myeloid leukemic cells may result in less sensitivity to various apoptotic stimuli. We examined whether human leukemia HL-60 cells differentiating by all-trans retinoic acid (ATRA) acquired resistance to the apoptogenic activity of two histone deacetylase (HDAC) inhibitors, butyrate and valproate. In undifferentiated cells, the cytotoxicity of both butyrate and valproate was associated with activation of the intrinsic apoptotic pathway since we observed dissipation of mitochondrial membrane potential, induction of caspase-9 and caspase-3 activities, appearance of sub-G1 DNA and loss of plasma membrane asymmetry and/or integrity. Both HDAC inhibitors were also able to induce accumulation of undifferentiated cells in the G0/G1 phase of the cell cycle. ATRA was found to enhance the apoptotic effect of both butyrate and valproate in undifferentiated cells. This aside, ATRA appeared to synergize with butyrate in the induction of the G0/G1 cell cycle arrest. In cells pretreated for 72 h with ATRA, butyrate and valproate in combination with ATRA induced lower dissipation of mitochondrial membrane potential and weaker apoptotic and/or necrotic changes in plasma membrane, whereas DNA fragmentation was not diminished compared to undifferentiated cells. Similar results were also obtained when butyrate or valproate were combined with another neutrophilic differentiation inducer, dimethyl sulfoxide. We conclude that neutrophilic differentiation modulates but does not abrogate the apoptotic response of HL-60 cells to butyrate and valproate, and nuclei are preferentially affected during apoptosis in differentiated cells.