1. A human recombinant homo-oligomeric 5-HT3 receptor (h5-HT(3A)) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole-cell recording configuration of the patch clamp technique. 2. 5-HT evoked transient inward currents (EC50 = 3.4 μM; Hill coefficient = 1.8) that were blocked by the 5-HT3 receptor antagonist ondansetron (IC50 = 103 pM) and by the non-selective agents metoclopramide (IC50 = 69 nM), cocaine (IC50 = 459 nM) and (+)-tubocurarine (IC50 = 2.8 μM). 3. 5-HT-induced currents rectified inwardly and reversed in sign (E(5-HT)) at a potential of -2.2 mV. N-Methyl-D-glucamine was finitely permeant. Permeability ratios P(Na)/P(Cs) and P(NMDG)/P(Cs) were 0.90 and 0.083, respectively. 4. Permeability towards divalent cations was assessed from measurements of E(5-HT) in media where Ca2+ and Mg2+ replaced Na+. P(Ca)/P(Cs) and P(Mg)/P(Cs) were calculated to be 1.00 and 0.61, respectively. 5. Single channel chord conductance (γ) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at -40 mV to 742 fS at -100 mV. 6. Reducing [Ca2+](o) from 2 to 0.1 mM caused an increase in the whole-cell current evoked by 5-HT. A concomitant reduction in [Mg2+](o) produced further potentiation. Fluctuation analysis indicates that a voltage-independent augmentation of γ contributes to this phenomenon. 7. The data indicate that homo-oligomeric receptors composed of h5-HT(3A) subunits form inwardly rectifying cation-selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.