The non-gastric H⁺/K⁺-ATPase ATP12A (ATP1AL1) is expressed in a broad variety of tissues, but the knowledge about its function is sparse. We found by RT-PCR and/or western blotting, measurements of intracellular pH, electron microprobe analysis, cell volume (CV) measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL60 cells, human polymorphonuclesr leucocytes (PMN), rat insulinoma Ins-1E cells and human pancreatic islets, as well as in normal and cancerous human prostate tissue. Activation of PMN by the chemotactic factor fMLP leads to cell swelling by activation of the Na⁺/H⁺-exchanger NHE1 and the H⁺/K⁺-ATPase and inhibition of either ion-transporter inhibits cell migration. Treatment of HL60 cells with low (1mM) concentrations of butyrate leads to differentiation towards the monocytic lineage whereas higher (5-10mM) concentrations induce apoptosis as assessed by flow cytometric determination of CD86 expression, CV, cell granularity, caspase activity, phosphatidyserine exposure on the outer plasma membrane leaflet, cell cycle analysis and cell proliferation. Similar, Ins-1Ecells undergo apoptosis upon treatment with the polyphenol resveratrol or by glucose starvation. Ins-1E cells and the fraction of HL60 cells bearing the common leucocyte antigen CD45 (CD45+) exhibit apparent apoptotic volume decrease (AVD). Untreated CD45+ cells have a greater volume than CD45- cells which also lack AVD. Transcriptional up-regulation of ATP12A is evident during both butyrate-induced differentiation and apoptosis in HL60 cells and during resveratrol-induced apoptosis in Ins-1E cells. Inhibition of the H⁺/K⁺-ATPase by the K⁺-competitive antagonist SCH28080 (10-100mM) leads per se to induction of apoptosis in differentiated HL60 cells and untreated Ins-1E cells and accelerates the time course of apoptosis in these cells if induced by high concentrations of butyrate or resveratrol, respectively. This is accompanied by acceleration and/or aggravation of AVD. Taken together, these results demonstrate that ATP12A is functionally active in PMN, differentiated HL60 cells and Ins-1E cells and that it plays a role during apoptosis at least in part by counteracting AVD. In line with this is the observation, that ATP12A gene expression is stage dependently up- or down regulated in cancerous tissue from human prostate cancer.