Palonosetron is a potent 5-HT3 receptor antagonist with a unique structure and some unusual properties. Here we explore the properties of palonosetron at heterologously expressed 5-HT3A and 5-HT3AB receptors. We used receptors expressed in HEK293 cells, and functionally analysed them using a membrane potential sensitive dye in a Flexstation, which revealed IC50s of 0.24 nM and 0.18 nM for 5-HT3A and 5-HT3AB receptors respectively. Radioligand binding studies with [3H]palonosetron revealed similar Kds: 0.34 nM for 5-HT3A and 0.15 nM for 5-HT3AB receptors. Kinetic studies showed palonosetron association and dissociation rates were slightly faster in 5-HT3AB than 5-HT3A receptors, and for both subtypes dissociation rates were ligand-dependent, with antagonists causing more rapid dissociation than agonists. Similar ligand effects were not observed for [3H]granisetron dissociation studies. These data support previous studies which show palonosetron has actions distinct to other 5-HT3 receptor antagonists, and the slow rates observed for agonist induced dissociation (t1/2 > 10 h) could at least partly explain the long duration of palonosetron effects in vivo.