Acid-sensing ion channels (ASICs), which belong to the epithelial sodium channel/degenerin (ENaC/DEG) family, are activated by extracellular protons and are inhibited by amiloride (AMI), an important pharmacological tool for studying all known members of ENaC/DEG.In this study, we reported that AMI paradoxically opened ASIC3 subtype channels at neutral pH and synergetically enhanced channel activation induced by mild acidosis (pH 7.2~6.8).The characteristic profile of AMI stimulation of ASIC3 channels was reminiscent of the channel activation by the newly identified nonproton ligand, 2-guanidine-4-methylquinazoline (GMQ).Using site-directed mutagenesis, we showed that ASIC3 activation by AMI, but not its inhibitory effect, was dependent on the integrity of the nonproton ligand sensing domain in ASIC3 channels.Furthermore, using covalent modification analyses, we provided strong evidence supporting that the nonproton ligand sensing domain is required for the stimulation of ASIC3 channels by AMI.These data reinforce the idea that ASICs can sense nonproton ligands in addition to protons.The results also raise a caution in the use of AMI for studying ASIC physiology and in the development of AMI-derived ASIC inhibitors for treating pain syndromes.