The results of a comprehensive structure-affinity relationship study on the effect of the quaternization (i.e., N-methylation) of structurally different ligands in the classes of tropane and quinuclidine derivatives are described. This study shows that the effects of the quaternization of the basic nitrogen of these 5-HT(3) receptor ligands appear to be strictly structure-dependent suggesting that different binding modes are operative at 5-HT(3) receptor binding site. The different effect of the quaternization of the basic nitrogen of structurally different ligands were rationalized in terms of the interaction with the receptor by means of the combined use of experimental techniques (X-ray diffraction and NMR studies) and computational simulation studies.