A computational model of the transmembrane domain of the human 5-HT4 receptorcomplexed with the GR113808 antagonist was constructed from the crystal structure of rhodopsin and the putative residues of the ligand-binding site, experimentally determined by site-directed mutagenesis. The recognition mode of GR113808 consist of: (i) the ionic interaction between the protonated amine and Asp3.32; (ii) the hydrogen bond between the carbonylic oxygen and Ser5.43; (iii) the hydrogen bond between the ether oxygen and Asn6.55; (iv) the hydrogen bond between the C-H groups adjacent to the protonated piperidine nitrogen and the pi electrons of Phe6.51; and (v) the pi-sigma aromatic-aromatic interaction between the indole ring and Phe6.52. This computational model offers structural indications about the role of Asp3.32, Ser5.43, Phe6.51, Phe6.52, and Asn6.55 in the experimental binding affinities. Asp3.32Asn mutation does not affect the binding of GR113808 because the loss of binding affinity from an ion pair to a charged hydrogen bond is compensated by the larger energetical penalty of Asp to disrupt its side chain environment in the ligand-free form, and the larger interaction between Phe6.51 and the piperidine ring of the ligand in the mutant receptor. In the Phe6.52Val mutant the indole ring of the ligand replaces the interaction with Phe6.52 by a similarly intense interaction with Tyr5.38, with no significant effect in the binding of GR113808. The mutation of Asn6.55 to Leu replaces the hydrogen bond of the ether oxygen of the ligand from Asn6.55 to Cys5.42, with a decrease of binding affinity that approximately equals the free energy difference between the SH...O and NH...O hydrogen bonds. Because these residues are also present in the other members of the neurotransmitter family of G protein-coupled receptors, these findings will also serve for our understanding of the binding of related ligands to their cognate receptors.