Nociceptors arising from the dorsal root ganglia (DRG) express acid-sensing ion channel-1 (ASIC1) subtypes to mediate the perception of inflammatory and neuropathic pain, and as such, these receptors are attractive targets for the development of analgesics for these painful conditions. Nevertheless, given that the human and rodent DRG differ considerably in subtype proportions of ASIC1 and that the pharmacological properties of rodent ASIC1 subtypes and their human homologues are distinct, ASIC1 inhibitors that demonstrate analgesic properties in rodents may not necessarily be effective in preventing pain in humans. In this study, we show that human embryonic kidney (HEK) 293 cells, which are routinely used as a cellular vehicle for the heterologous expression and pharmacological characterization of receptors and ion channels, natively transcribe the human homologues of ASIC1a and ASIC1b at similar proportions to those found in the human DRG. Importantly, HEK 293 ASIC1 is sensitive to inhibition by amiloride, ethylisopropyl amiloride, and the snake toxin mambalgin-1, but insensitive to inhibition by the ASIC1a inhibitor psalmotoxin-1 when applied at a physiological conditioning pH. Given that the human DRG transcribes the same set of ASIC1 subtypes as HEK 293 cells, our data support the notion that mambalgin-1 may be effective against acid pain sensation in humans. Moreover, our data suggest that the HEK 293 cell line may be a suitable tool for pharmacological screening and characterization of heteromeric human ASIC1.