The second-stage dissociation constants of N,N-bis(2-hydroxyethyl)glycine (bicine) were determined at 25.00 ± 0.02 °C by potentiometric pH titration in pure water and different mixed solvent mixtures (methanol, ethanol, -dimethylformamide, dimethyl sulfoxide, acetone, and dioxane). It is observed that the pKa2 value is slightly influenced as the solvent is enriched in methanol or ethanol and remains practically constant in the presence of different amounts of DMF and DMSO. A pronounced change in the pKa2 value 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 imino group (-NH+). Other solvent effects such as solvent basicity and different stabilization of the conjugate acid free base by hydrogen-bonding interactions in aquo-organic solvent media relative to pure aqueous media as well as proton-solvent interaction play an important role in the acid dissociation equilibrium. © 1994, American Chemical Society. All rights reserved.