Myelomonocytic HL60 cells are a proper 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 10 mM butyrate induces apoptosis within 48 h as assessed by flow cytometry (7AAD nuclear staining/phosphatidylserine exposure; activation of caspases) whereas a lower concentration of butyrate (1 mM) elicits the expression of CD86 within 48-72 h, indicating differentiation mainly towards the monocytic lineage. In cells treated with 1mM butyrate the number of apoptotic cells is not different compared to untreated cells, whereas inhibition of the non-gastric H⁺/K⁺-ATPase ATP12A by 100 mM SCH28080 in 1mM butyrate-treated HL60 cells is followed by a significantly accelerated apoptosis within 48-72 h. Similar apoptotic volume decrease (AVD) in 1 mM butyrate-treated HL60 cells is significantly more pronounced in the presence of the inhibitor. In addition HL60 cells show a significant up-regulation of ATP12A mRNA within 48 h of butyrate treatment (1 mM) as examined by quantitative PCR. Thus inhibition of ATP12A by SCH28080 fosters apoptosis in cells differentiated by 1 mM butyrate. In conclusion these data suggest an anti-apoptotic function of the non-gastric H⁺/K⁺-ATPase ATP12A in HL60 cells.