The second stage dissociation constants of N-[Tris-(hydroxymethyl)methyl]-2-aminoethane sulphonic acid (TES) were determined at 25±0.1 °C by potentiometric pH-titration in different mixed solvent mixtures. The organic solvents used were methanol, ethanol, dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone, and dioxane. The ionization of TES depends on both the proportion and the nature of the organic co-solvent used. It was observed that the pKa2 value is slightly influenced as the solvent is enriched in methanol, ethanol and remains practically constant in presence of different amounts of DMF and DMSO. A pronounced change in the pKa2 is observed as the solvent is enriched in acetone or dioxane. These results are discussed in terms of various solvent characteristics. It is concluded that the electrostatic effect has only a relatively small influence on the dissociation equilibrium of the cationic group - {Mathematical expression}H2. Other solvent effects such as solvent basicity, different stabilization of the conjugate acid free base by hydrogen bonding interactions in aquo-organic solvents media relative to pure aqueous media as well as proton-solvent interaction play an important role in the acid dissociation equilibrium. © 1994 Springer-Verlag.