TASK-2 (KCNK5 or K2P5.1) is a background K+ channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pHo) TASK-2 is gated open by intracellular alkalinization. Standard whole-cell patch-clamp recordings and an intracellular pH-clamp method based on the distribution of the weak acid acetate were used to study the modulation of TASK-2 expressed in HEK-293 cells by intracellular pH (pHi). There were significant increases in TASK-2 currents measured at 0mV upon alkalinization from 7.0 to 9.0. Fits of a Hill equation to the data gave average pK1/2 and nH values of 8.0 ± 0.07 and 0.9 ± 0.04 (mean ± SEM, n=7). Equivalent figures for TASK-2-R224A, a pHo-insensitive mutant (Niemeyer et al., 2007), were 7.7 ± 0.22 and 0.9 ± 0.16 (n=5). Point mutations of various titratable residues facing the intracellular aspect of the channel led to the identification of K245 as a residue whose neutralization by mutation to A or C removed pHi-dependence from TASK-2. Mutation of K245 to H preserved pHi-sensitivity but shifted pK1/2 to 6.8 ± 0.06 (n=4), consistent with a role of K245 as a pHi-sensor. We have found that it is not possible to overcome closure by extracellular acidification by means of intracellular alkalinization and that mutant TASK-2-R224A that lacks sensitivity to pHo has normal pHi-dependent gating. Also, increasing the extracellular K+ concentration acid-shifts pHo-activity curve of TASK-2 yet does not affect pHi-gating of TASK-2 and whilst pHo modulation of TASK-2 is voltage-dependent pHi-gating was not altered by membrane potential. These results suggest that pHo, which controls a selectivity filter external gate, and pHi act at different gating processes to open and close TASK-2 channels. We conclude that intracellular pH, together with pHo, is a critical determinant of TASK-2 activity and therefore of its physiological function. Our data also provide a way to link extracellular CO2 levels (and therefore intracellular pH) with the activity of TASK-2, a requirement to fulfil its recently proposed role (Gestreau et al., 2010) in the control of firing of central chemosensing neurons.