Hydrolysis of α^4,α^5-O-isopropylidene pyridoxine (4,5-IPP) and factors influencing on the hydrolysis were studied. Solubility of 4,5-IPP in water was better than that of α^4,3-O-isopropylidene pyridoxine (3,4-IPP) but vice versa in organic solvents. In buffer solutions of a constant pH between 1 and 6,the hydrolysis of 4,5-IPP obeyed an apparent first-order kinetics and was faster than that of 3,4-IPP. It was found that ionic strength, buffer concentration and buffer constituents did not influence the hydrolysis rate but concentration of ethanol affected on the hydrolysis. Under these conditions, rearrangement from 4,5-IPP to 3,4-IPP did not occur.