Palonosetron (Aloxi) is a potent second generation 5-HT 3 receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT 3 receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr 73 , Phe 130 , Ser 163 , and Asp 165 ) and in the 5-HT3B receptor subunit (His 73 , Phe 130 , Glu 170 , and Tyr 143 ) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT 3 A) and heteromeric (5-HT 3 AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT 3 A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking.