The activation of α9α10 nicotinic receptors in cochlear hair cells can ameliorate acoustic trauma. Therefore, maximized α9α10 receptor activation may favor the prevention of noise-induced hearing loss. Hence, understanding the conditions in which α9α10 receptors reach maximum activation could have a potential therapeutic use in noise-induced hearing loss. In this work we aimed to characterize how extracellular Ca2+ affects the activation of recombinant α9α10 receptors expressed in X. laevis oocytes under two-electrode voltage-clamp. Previous work from our lab suggests that the homomeric α9 receptor reaches maximum activation at trace concentrations of extracellular Ca2+ and is blocked by higher concentrations (IC50 = 100±10 μM, n = 3). We now show that the α9α10 receptor, as opposed to homomeric α9, reaches its lowest maximal currents (at saturating acetylcholine) at trace levels of extracellular Ca2+ (50±15% of maximal currents at physiological 1.8 mM Ca2+; n = 4) and its maximal activation at Ca2+ concentrations close to 0.1mM (260±55% of maximal currents at 1.8 mM Ca2+; n = 5). Furthermore, increasing extracellular Ca2+ concentrations reduces the receptor’s EC50 dose dependently (trace Ca2+: EC50 = 54±4 μM, n = 7; 1.8 mM Ca2+: EC50 = 13±2 μM, n = 8). This differential dependency upon extracellular Ca2+ suggests that the α10 subunit provides the structural determinants. In order to delineate these structural determinants, we constructed chimeric α10-α9 subunits. Receptors with chimeric α10 subunits bearing the α9 extracellular and the α10 transmembrane and intracellular domains reached maximal activation at trace levels of extracelular Ca2+ (190±20% of maximal currents at 1.8 mM Ca2+; n = 5). The inclusion of α9 transmembrane regions facing the extracellular domain (the TM2-TM3 loop and extracellular segment of TM1) in the chimeras further boosted the activation at trace extracellular Ca2+ (390±40% of maximal currents at 1.8 mM Ca2+; n = 4). Altogether, our results suggest that extracellular Ca2+ greatly affects α9α10 receptor activation and that the structural determinants of this effect are located at the interphase between extracellular and transmembrane domains of the α10 subunits, a region known to be responsible for channel gating.