Myelomonocytic HL60 cells are a suitable model for multidirectional (monocytic, eosinophilic and granulocytic) differentiation. In this work we investigate the relationship between induction of differentiation and of apoptosis in HL60 cells. Treatment of the cells with 5–10mM butyrate induces apoptosis within 24h as assessed by flow cytometry (7AAD nuclear staining/phosphatidylserine exposure; activation of caspases) whereas a lower concentration of butyrate (1mM) elicits the expression of the monocytic marker CD86 within 48–72h. In cells treated with 1mM butyrate the number of apoptotic cells is not different compared to untreated cells. In 1mM butyrate-treated cells inhibition of the non-gastric H⁺/K⁺-ATPase ATP12A by 100mM SCH28080 is followed by intracellular acidification and significantly accelerated apoptosis. Similar apoptotic volume decrease (AVD) is significantly more pronounced in the presence of the inhibitor. In addition HL60 cells show a significant up-regulation of ATP12A mRNA within 48h of butyrate treatment (1mM) as examined by quantitative PCR. Thus inhibition of ATP12A by SCH28080 fosters apoptosis in cells differentiated by 1mM butyrate. In conclusion these data suggest an anti-apoptotic function of the non-gastric H⁺/K⁺-ATPase ATP12A in HL60 cells.