The TRPM3 gene encodes for many different proteins due to alternative splicing. One of the splice sites is situated in the ion-conducting pore region. Splicing at this site alters the biophysical properties of the pore and changes the relative permeability of the channels for divalent cations by at least one hundred fold. To better understand the permeation process through the channels with higher divalent permeability (called TRPM3a2), we investigated the relative permeability of a variety of mono- and divalent cations by measuring bi-ionic reversal potentials after agonist stimulation. Probing the maximal diameter of the pore with amines of various sizes, we estimated it to be rather large, approx. 0.7 nm. Nevertheless, the channel is preferentially permeable for divalent cations (Ca²⁺, Mg²⁺ and Mn²⁺ were approx. 10 times and Ba²⁺ 4 times more permeable than Cs⁺), but it discriminates only poorly between monovalent inorganic cations (less than 2 fold differences in permeability ratios). However, the amplitude of the current carried by monovalent inorganic cations varied greatly, with Na⁺ having the lowest absolute permeability. These data indicate that TRPM3a2 channels possess at least 2 binding sites for cations, one preferring divalent, the other monovalent cations. The latter is likely to be a strong-field-strength site.