The 5-hydroxytryptamine type 3 (5-HT3) receptor is a transmitter-gated ion channel mediating neuronal excitation. The receptor native to neurons, or as a homopentameric assembly of 5-HT(3A) receptor subunits, displays a species-dependent pharmacology exemplified by a 1800-fold difference in the potency of (+)-tubocurarine [(+)-Tc] as an antagonist of the current response mediated by mouse and human receptor orthologs. Here, we attempt to identify amino acid residues involved in binding (+)-Tc by use of chimeric and mutant 5-HT(3A) subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5- HT(3A) (m5-HT(3A)) subunit by that of the human ortholog and vice versa exchanged the differential potency of (+)-Tc, demonstrating the ligand binding site to be contained wholly within this region. Mutagenesis of multiple amino acid residues within a putative binding domain that exchanged non-conserved residues between mouse and human receptors shifted the apparent affinity of (+)-Tc in a reciprocal manner. The magnitude of the shift increased with the number of residues (3, 5, or 7) exchanged, with septuple mutations of m5-HT(3A) and human 5-HT(3A) subunits producing a 161-fold decrease and 53-fold increase in the apparent affinity of (+)-Tc, respectively. The effect of point mutations was generally modest, the exception being m5-HT(3A) D206E, which produced a 9-fold decrease in apparent affinity. We conclude that multiple amino acids within a binding loop of human and mouse 5-HT(3A) subunits influence the potency of (+)-Tc.