The serotonin 5-HT₃ receptor native to rat hippocampal CA1 stratum radiatum interneurons is blocked by Ca²⁺ ions in a dose- and voltage-dependent manner, which is reflected by a region of negative slope conductance in the I-V curve. The steep dependence on the extracellar Ca²⁺ concentration suggests that the channel contains more than one binding site for Ca²⁺. A 3 barrier - 2 site model, based on Eyring rate theory, was used to describe the I-V curves. When extra- and intracellular K⁺ and Cs⁺ were substituted with Na⁺, the I-V curves were accurately fit by the model, unlike the I-V curves recorded under standard ionic conditions. This suggests that the K⁺ and Cs⁺ permeabilities are small as compared to that of Na⁺. The distribution of the energy barriers and binding sites for Ca²⁺ and Na⁺ showed that the binding sites are located at approximately the 13' and the -4' position in the ion channel. The model predicts that at large hyperpolarized membrane potentials (more negative than -120 mV), the fractional Ca²⁺ current amounts to ~1% of the total ion current. However, at physiologically relevant membrane potentials, the fractional Ca²⁺ current is smaller (<0.1%) and the relative Ca²⁺ permeability (P_Ca/P_Na) is estimated to be 0.10 at -60 mV.