Recently, we have identified matrix pH as a key regulator of mitochondrial energy metabolism in the β-cell (Wiederkehr et al. EMBO J. (2009) 28(4):417-28). Under resting glucose conditions matrix pH in β-cell mitochondria is unusually low (pH 7.25). Following nutrient stimulation of rat islets matrix alkalinisation occurs raising matrix pH to 7.7 a value close to those determined in other cell types (pH 7.8-8.1). Alkalinisation follows a very similar time-course as net increases of the cytosolic ATP levels and is associated with a 2.5-fold elevation of the mitochondrial ATP synthesis rate. Suppression of this alkalinisation using nigericin almost completely abolishes mitochondrial ATP synthesis in a permeabilised cell system. Our working hypothesis is that low mitochondrial pH maintains the β-cell mitochondria in an inactive state, whereas nutrient stimulation favors alkalinisation and full activation of mitochondrial energy metabolism, resulting in mitochondrial signal generation and insulin granule exocytosis.