The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapid delayed rectifier potassium current (IKr), contributing to the repolarisation of ventricles. The hERG-mediated current is modulated by extracellular acidosis that occurs during certain pathological heart conditions such as ischemia (Du et al. 2010; Van Slyke et al. 2012; Jiang et al. 1999; Anumonwo et al. 1999; Bett & Rasmusson 2003). We have studied the effects of extracellular acidosis on macroscopic wild-type (WT) hERG channels, with the aim of resolving the identity of the amino acid(s) that interact with extracellular protons. Whole-cell patch clamp measurements of IhERG were made at room temperature using either HEK-293 or CHO cells expressing hERG, with the effects of extracellular acidosis (pH 6.3 and 5.5) determined under voltage clamp. In addition, experiments were conducted to derive a pKa for a number of IhERG properties. Data are presented as mean ± SEM. Extracellular acidosis (to pH 6.3) caused a significant positive shift in activation of IhERG, an acceleration in deactivation, as well as a reduction in macroscopic current conductance from 0.34 ± 0.04 nS/pF at pHe 7.4 to 0.22 ± 0.04 nS/pF at pHe 6.3 (n=8 cells; P = 0.0003, two-tailed paired t-test). A further reduction was seen in IhERG conductance at pHe 5.5 (from 0.36 ± 0.04 nS/pF at pHe 7.4 to 0.10 ± 0.01 nS/pF at pHe 5.5; n=8 cells; P < 0.0001, two-tailed paired t-test). Determination of the sensitivity of different current properties to pH revealed that more than one distinct pKa value were apparent for different processes measured. Thus, sensitivity of the end-pulse current to pH gave a pKa of 6.04 ± 0.09 (n=8 cells) while the amplitude of the peak tail current was sensitive to pH with a pKa of 5.61 ± 0.21 (n=8 cells). Current deactivation was best described by two exponentials, each displaying a different sensitivity to pH: the fast component of deactivation had a pKa of 6.53 ± 0.07 (n=8 cells), while the slow component exhibited a pKa of 7.08 ± 0.35 (n=8 cells) These data illustrate that more than one site of protonation might be responsible for the sensitivity of the hERG to extracellular acidosis (Bett & Rasmusson 2003). Ongoing mutagenesis work is targeting a cluster of residues located in the S5 and pore-loop region of the hERG channels to test for attenuation of sensitivity to protons.