Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (I-1,2,3,4,5,6-P6). A number of phytases with varying specificities, properties, and localizations hydrolyze phytic acid present in cells. The specificity of hydrolysis of phytic acid by alkaline phytase from lily (Lilium longiflorum L.) pollen is described. Structures of the intermediate inositol phosphates and the final product were established by a variety of nuclear magnetic resonance techniques (1H-, 31P-, and 31P-1H-detected multiple quantum coherence spectroscopy, and total correlation spectroscopy). On the basis of the structures identified we have proposed a scheme of hydrolysis of phytic acid. Initial hydrolysis of the phosphate ester occurs at the D-5 position of phytic acid to yield the symmetrical I-1,2,3,4,6-P5. The two subsequent dephosphorylations occur adjacent to the D-5 hydroxyl group to yield I-1,2,3-P3 as the final product. Alkaline phytase differs from other phytases in the specificity of hydrolysis of phosphate esters on the inositol ring, its high substrate specificity for phytic acid, and biochemical properties such as susceptibility to activation by calcium and inhibition by fluoride. The physiological significance of alkaline phytase and the biological role of I-1,2,3-P3 remain to be identified.