The 5-HT(3) receptor is a member of the Cys-loop family of ligand-gated ion channels. The extracellular domains of these proteins contain six amino acid loops (A - F) which converge to form the ligand binding site. In this study we have mutated 21 residues in or close to the 5-HT(3) receptor F-loop (Ile-192 to Gly-212) to Ala or to a residue with similar chemical properties. Mutant receptors were expressed in HEK293 cells and binding affinity measured using [(3)H]granisetron. Two regions displayed increases in binding affinity when mutated to Ala (Ile-192 - Arg-196 and Asp-204 - Ser-206), but only one region was sensitive when mutated to chemically similar residues (Ile192 - Val201). Homology modeling using AChBP crystal structures with a variety of different bound ligands suggests there may be distinct movements of Trp-195 and Asp-204 upon ligand binding, indicating that these residues and their immediate neighbors have the ability to interact differently with different ligands. The models suggest predominantly lateral movement around Asp-204 and rotational movement around Trp-195, indicating the former is in a more flexible region. Overall our results are consistent with a flexible 5-HT(3) receptor F-loop with two regions that have specific but distinct roles in ligand binding.