Site-directed mutagenesis and molecular modeling were used to investigate the molecular interactions involved in ligand binding to, and activation of, the rat 5-hydroxytryptamine(2A) (5-HT(2A)) serotonin (5-HT) receptor. Based on previous modeling studies utilizing molecular mechanics energy calculations and molecular dynamics simulations, four sites (S239[5.43], F240[5.44], F243[5.47], and F244[5.48]) in transmembrane region V were selected, each predicted to contribute to agonist and/or antagonist binding. The F243A mutation increased the affinity of (+/-)4-iodo-2, 5-dimethoxyphenylisopropylamine, decreased the binding of alpha-methyl-5HT, N-omega-methyl-5HT, ketanserin, ritanserin, and spiperone and had no effect on the binding of 5-HT and 5-methyl-N, N-dimethyltryptamine. The F240A mutant had no effect on the binding of any of the ligands tested, whereas F244A caused an agonist-specific decrease in binding affinity (3- to 10-fold). S239A caused a 6- to 13-fold decrease in tryptamine-binding affinity and a 5-fold increase in affinity of 4-iodo-2, 5-dimethoxyphenylisopropylamine. A subset of the agonists used in binding studies were used to determine the efficacies and potencies of these mutants to activate phosphoinositide hydrolysis. The F243A and F244A mutations reduced agonist stimulated phosphoinositide hydrolysis, whereas the S239A and F240A mutations had no effect. There was little correlation between agonist binding and second messenger production. Furthermore, molecular dynamics simulations, considering these data, produced ligand-bound structures utilizing substantially different bonding interactions even among structurally similar ligands (differing by as little as one methyl group). Taken together, these results suggest that relatively minor changes in either receptor or ligand structure can produce drastic and unpredictable changes in both binding interactions and 5-HT(2A) receptor activation. Thus, our finding may have major implications for the future and feasibility of receptor structure-based drug design.