The purpose of the present study was to examine 5-hydroxytryptamine (5-HT)_2A-receptor sarpogrelate interactions by site-directed mutagenesis. Based on molecular modeling studies, aspartic acid (Asp)155[3.32] and tryptophan (Trp)151[3.28] in transmembrane helix (TMH) III and Trp336[6.48] in TMH VI of the 5-HT_2A receptor were found to interact with sarpogrelate. All of these residues were mutated to alanine (Ala). The Asp3.32Ala mutant did not exhibit any affinity for [³H]ketanserin, whereas the Trp3.28Ala mutant showed a markedly decrease in the binding affinity for [³H]ketanserin (K_d >10,000 nM). Therefore, it was not possible to find any sarpogrelate affinity to the mutants using [³H]ketanserin. The mutation also abolished agonist-stimulated formation of [³H]inositol phosphates (IP) in both cases. On the other hand, the Trp6.48Ala mutant showed reduced binding affinity for [³H]ketanserin (K_d 2.0 nM vs 0.8 nM for the wild-type receptor) and had reduced affinity for sarpogrelate (pK_i value of 5.71 vs 9.06 for the wild-type receptor). The Trp6.48Ala mutant also showed the greatest decrease in sensitivity to sarpogrelate (pK_b value 8.81 for wild-type and 5.11 for mutant) in inhibiting agonist-stimulated IP formation. These results provide direct evidence that Asp3.32, Trp3.28, and less importantly, Trp6.48 are responsible for the interaction between the 5-HT_2A receptor and sarpogrelate. In addition, these results support the findings obtained from molecular modeling studies.