Proton receptors of the acid-sensing ion channel (ASIC) family are expressed in sensory neurons and thus could play a critical role in the detection of noxious acidosis. To investigate the subunit composition of native ASICs in peripheral and central neurons, we co-injected human as well as rodent ASIC2a and ASIC3 subunits in Xenopus oocytes. The amplitudes of acid-induced biphasic responses mediated by co-expressed ASIC2a and ASIC3 subunits were much larger (as much as 20-fold) than the currents mediated by the respective homomers, clearly indicating functional association. The reversal potential of the ASIC2a+3 current (>/=+20 mV) reflected a cationic current mainly selective for sodium. The sensitivity to pH or amiloride of single versus co-expressed ASIC subunits was not significantly different; however, gadolinium ions inhibited ASIC3 and ASIC2a+3 responses with much higher potency (IC(50) approximately 40 microm) than the ASIC2a response (IC(50) >/=1 mm). Biochemical interaction between ASIC2a and ASIC3 subunits was demonstrated by co-purification from transfected human embryonic kidney (HEK293) cells and Xenopus oocytes. Our in situ hybridization data showed that rat ASIC2a and ASIC3 transcripts are co-localized centrally, whereas reverse transcription-polymerase chain reaction data led us to detect co-expression of human ASIC2a and ASIC3 subunits in trigeminal sensory ganglia, brain, and testis where they might co-assemble into a novel subtype of proton-gated channels sensitive to gadolinium.