The FlexStation is a 96 or 384 fluorescent plate reader with the capability of adding solutions during readings; it therefore has the potential to provide high throughput analyses of the functional characteristics of neurotransmitter-gated ion channels that can be examined using changes in fluorescence. The 5-HT3 receptor is one such protein, as its activation results in a change in membrane potential due to the opening of a Ca2+ -permeant, cation-selective channel; it can therefore be studied using both Ca2+ - and membrane potential-sensitive fluorescent dyes. Here we have used the FlexStation to examine the function of recombinant 5-HT3 receptors expressed in HEK293 cells using both these classes of dye. The results show that the pharmacological characteristics of the receptor obtained using the FlexStation is similar to those reported using other functional methods, although caution must be applied when using the membrane potential dye as large changes in membrane potential can yield inaccurate EC50s. Modifying the constituents of the buffer, however, so that the change in membrane potential was reduced, yielded EC50 values that were similar to previously reported data. We conclude that the FlexStation is a useful tool for high throughput studies when examining the function of neurotransmitter receptors that result in either a change in Ca2+ concentration or membrane potential.