Palonosetron is a 5-HT(3) receptor antagonist that has demonstrated superiority in preventing both acute and delayed emesis when compared to older first generation 5-HT(3) receptor antagonists. The objective of this work was to determine if palonosetron exhibits unique molecular interactions with the 5-HT(3) receptor that could provide a scientific rationale for observed clinical efficacy differences. Previously, we showed that palonosetron exhibits allosteric binding and positive cooperativity to the 5-HT(3) receptor in contrast to ondansetron and granisetron which exhibit simple bimolecular binding. The present work shows, through several independent experiments, that palonosetron uniquely triggers 5-HT(3) receptor internalization and induces prolonged inhibition of receptor function. After 24h incubation followed by dissociation conditions, [(3)H]palonosetron remained associated with whole cells but not to cell-free membranes (P<0.001). [(3)H]Palonosetron's binding to cells was resistant to both protease and acid treatments designed to denature cell surface proteins suggesting that the receptor complex was inside the cells rather than at the surface. Cells pretreated with unlabeled palonosetron subsequently exhibited reduced cell surface 5-HT(3) receptor binding. Palonosetron-triggered receptor internalization was visualized by confocal fluorescence microscopy using cells transfected with 5-HT(3) receptor fused to enhanced cyan fluorescent protein. In contrast, granisetron and ondansetron showed minimal to no effect on receptor internalization or prolonged inhibition of receptor function. These experiments may provide a pharmacological basis for differences noted in published clinical trials comparing palonosetron to other 5-HT(3) receptor antagonists.