1. The pharmacological and biophysical properties of a recombinant 5-HT3 receptor have been studied by use of patch-clamp techniques applied to HEK 293 cells stably transfected with the murine 5-HT3 R-A cDNA. 2. At a holding potential of -60 mV, 77% of cells investigated responded to ionophoretically applied 5-HT with an inward current. Such currents were unaffected by methysergide (1 microM), or ketanserin (1 microM), but were antagonized in a concentration-dependent and reversible manner by the selective 5-HT3 receptor antagonist, ondansetron (IC50 = 440 pM) and the non-selective antagonists (+)-tubocurarine (IC50 = 1.8 nM) and metoclopramide (IC50 50 nM). 3. The 5-HT-induced current reversed in sign (E5-HT) at approximately -2mV and exhibited inward rectification. The influence of extra- and intracellular ion substitutions upon E5-HT indicates the 5-HT-evoked current to be mainly mediated by a mixed monovalent cation conductance. 4. Calcium and magnesium (0.1-10 nM) produced a concentration-dependent, voltage-independent, inhibition of the 5-HT-induced response. Zinc (0.3-300 microM) exerted a biphasic effect with low concentrations enhancing, and high concentrations depressing, the 5-HT-evoked current. 5. Fluctuation analysis of inward currents evoked by a low (1 microM) concentration of 5-HT suggests the current to be mediated by the opening of channels with a conductance of 420 fS. 6. The pharmacological and biophysical properties of the 5-HT3 R-A are similar to those previously described for 5-HT3 receptors native to murine neuroblastoma cell lines, with the exception that the function of the recombinant receptor was enhanced by low concentrations of zinc. This observation suggests that the properties of the native receptor are not completely represented by the 5-HT3 R-A subunit alone.