Potassium channels encoded by the human Ether-à-go-go-Related Gene (hERG) are believed to underpin the rapid delayed rectifier K+ current (IKr), which plays a key role in ventricular repolarisation in the heart. Acidosis occurs in a number of pathological situations such as cardiac ischemia and is known to modulate macroscopic hERG channel current, IhERG (e.g. Bérubé et al. 1999; Terai et al. 2000; Bett & Rasmusson 2003; Du et al. 2010; Van Slyke et al. 2012). We have studied the effect of extracellular acidosis on single wild-type hERG channels, to identify actions that may underpin reported effects of acidosis on macroscopic IhERG. Whole-cell and cell-attached patch clamp measurements of IhERG from HEK-293 cells stably expressing hERG channels were made at room temperature. For macroscopic IhERG recordings, the extracellular superfusate was acidified whereas for cell-attached recordings unpaired comparisons were made between recordings made with pipette solutions of pH 7.4 and 6.3. Data are presented as mean ± SEM. Macroscopic IhERG responded to a reduction in pHe by a modest positive shift in activation, accelerated deactivation and reduced whole cell IhERG conductance (from 0.69 ± 0.12 nS/pF at pHe 7.4 to 0.46 ± 0.09 nS/pF at pHe 6.3; n=8 cells; P < 0.001, two-tailed paired t-test). Single channel recordings were made with symmetrical K+ concentrations of 140mM. Current measurements were made at a series of repolarization voltages following a depolarizing command to +40 mV. A reduction in pHe caused a reduction in single channel amplitude with a mean amplitude, at -100mV, of 1.11 ± 0.03 pA at pH 7.4 (n=8 cells) compared to 0.93 ± 0.08 pA at pH 6.3 (n=7 cells). Slope conductance values derived from current-voltage relations between -120 and -40 mV were 11.31 ± 0.17 pS for pH 7.4 (n=8 cells) and 9.39 ± 0.44 pS for pH 6.3 (n=7 cells; P < 0.01, two-tailed unpaired t-test with Welch’s correction) respectively. Thus, a reduction in single channel conductance contributes to the attenuation of macroscopic IhERG by extracellular acidosis.