The protonation process of the individual functional groups of four analogues of (±)-myo-inositol 1,4,5-tris(phosphate) [Ins(1,4,5)P3], modified at position 6, were studied by 31P- and 1H-NMR titration experiments in order to gain insight into the reasons for the particular importance of OH6 for the binding of Ins(1,4,5)P3 to its receptor. The fluorinated derivative (2) was studied to evaluate the H-bond donor or acceptor ability of OH6. Compounds 3 and 4 should explain the presence and the configuration of OH6, respectively, whereas the amino analogue (5) was considered to delineate the effect of a positively charged group of about the same size as OH6 on the vicinal phosphates P1 and P5. The 31P-NMR curves look alike for compounds 2–4 and differ from that of Ins(1,4,5)P3 , as the initial downfield shift of P1 upon protonation is no longer observed. In the 1H-NMR titration curves, the wrongway shift found for H2 of Ins(1,4,5)P3 is observed for H6 in compounds 3 and 4. In addition, both phosphorus and proton resonances of compound 5 are influenced by the protonation of the neighbouring NH2 group. By considering the protonation constants it is shown that the log K1 values decrease in the order 6-F-Ins(1,4,5)P3>6-deoxy-Ins(1,4,5)P3>Ins(1,4,5)P3> epi-Ins(1,4,5)P3>6-NH2-Ins(1,4,5)P3, indicating that the substitution effect is the consequence of the lipophilicity of the substituents, the basicity order following the order of the hydrophobic π constants. Consideration of the microprotonation constants, calculated for all the studied compounds, leads to the conclusion that hydration effects and hydrogen bond donor ability of the equatorial OH6 hydroxyl should mainly account for the differences observed between compounds 2–4 and Ins(1,4,5)P3. The amino derivative 5 remains in a zwitterionic form over almost the entire pH range studied (2.5<pH<11.5). Through the presence of an intramolecular hydrogen-bonded ion pair interaction the basicity of all three phosphates is decreased whereas the basicity of the amine increases. The strength of the hydrogen-bonded ion pair appears to be of the same order of magnitude as that of the recently published monoammonium phosphate complexes.