AbstractDrug-receptor interaction theory predicts that proportional receptor occupancy is a function of ligand concentration as defined by a ligand-receptor affinity constant, and is independent of receptor density. However, we previously observed that the EC₅₀ of 5-HT reduced as the density of 5-HT₃ receptors increased, suggesting an effect of receptor density on occupancy. The current study was designed to maximise variability in experimentally observed currents and confirm this apparent contradiction prospectively. Xenopus oocytes were injected with RNA encoding 5-HT₃A receptors under conditions designed to achieve varying receptor expression levels and 5-HT-evoked currents measured using two electrode voltage clamp. Results from 99 oocytes showed that as the maximal peak current increased from 0.05 µA to 12.1 µA there was a 3.7-fold reduction in EC₅₀. Since occupancy and conductance are directly related in this system, this indicates that for a given concentration of 5-HT, proportional occupancy increases with increased receptor density. We conclude that normalising data masks this correlation, and can result in reduced accuracy of pharmacological measurements. We propose a mechanistic explanation for our observations.