Phytic acid ( myo -inositol hexakisphosphate, Ins P 6 ) hydrolysis by Bacillus phytase (PhyC) was studied. The enzyme hydrolyses only three phosphates from phytic acid. Moreover, the enzyme seems to prefer the hydrolysis of every second phosphate over that of adjacent ones. Furthermore, it is very likely that the enzyme has two alternative pathways for the hydrolysis of phytic acid, resulting in two different myo -inositol trisphosphate end products: Ins(2,4,6) P 3 and Ins(1,3,5) P 3 . These results, together with inhibition studies with fluoride, vanadate, substrate and a substrate analogue, indicate a reaction mechanism different from that of other phytases. By combining the data presented in this study with (1) structural information obtained from the crystal structure of Bacillus amyloliquefaciens phytase [Ha, Oh, Shin, Kim, Oh, Kim, Choi and Oh (2000) Nat. Struct. Biol. 7 , 147Ő153], and (2) computer-modelling analyses of enzymeŐsubstrate complexes, a novel mode of phytic acid hydrolysis is proposed.