The serotonin 5-HT 3 receptor (5-HT 3 R) is a member of the cys-loop ligand-gated ion channel family. We have used the combination of site-directed mutagenesis, homology modeling of the 5-HT 3 R extracellular domain, and ligand docking simulations as a way to map the architecture of the 5-HT 3 R ligand binding domain. Mutation of Phe226 in loop C of the binding site to tyrosine (F226Y) has no effect on the apparent affinity of the competitive antagonist d-tubocurarine (dTC) for the receptor. On the other hand, replacement of Asn128 in loop A of the binding site with alanine (N128A) increases the apparent affinity of dTC by approximately 10-fold. Double-mutant cycle analysis employing a panel of dTC analogs with substitutions at various positions to identify specific points of interactions between the dTC analogs and Asn128 suggests that Asn128 makes a direct interaction with the 2ЈN of dTC. Molecular modeling of the 5-HT 3 R extracellular domain using the antagonist-bound conformation of the Aplysia californica acetylcholine binding protein as a template followed by ligand docking simulations produces two classes of structures of the 5-HT 3 R/dTC complex; only one of these has the 2ЈN of dTC positioned at Asn128 and thus is consistent with the data from this study and previously published data. The use of the rigid dTC analogs as "molecular rulers" in conjunction with double-mutant cycle analysis of mutant receptors can allow the spatial mapping of the position of various residues in the ligand-binding site.The serotonin type 3 receptor (5-HT 3 R) is a member of the cys-loop ligand-gated ion channel gene family, which includes the muscle and neuronal nicotinic acetylcholine receptors (AChR), the glycine receptor, and the GABA type A receptor (Connolly and Wafford, 2004;Lester et al., 2004). Two different subunits, termed 5-HT 3A and 5-HT 3B , have been shown to be present in functional 5-HT 3 Rs (Reeves and Lummis, 2002). In addition, several other putative 5-HT 3 R genes have been described, but whether or not they play a role in 5-HT 3 R-mediated processes is unknown at present (Karnovsky et al., 2003). The 5-HT 3A subunit alone can form functional receptors with the appropriate pharmacological properties when expressed in Xenopus laevis oocytes or mammalian cells. However, receptors comprising only the 5-HT 3A subunit have single-channel conductances in the subpicosiemens range, whereas those containing both the 5-HT 3A and 5-HT 3B subunits have conductances in the 10-to 30-pS range (Davies et al., 1999;Dubin et al., 1999;Hanna et al., 2000), similar to that observed in neurons from the peripheral nervous system (Derkach et al., 1989;Yang et al., 1992). The reduction in single-channel conductance of homomeric 5-HT 3A Rs is now know to be due to the presence of three arginine residues in the cytoplasmic domain, and removal of these three positive charges results in a single-channel conductance on the order of 25 pS (Kelley et al., 2003). Analysis of the expression patterns of the 5-HT 3A and 5-HT 3B su...