The pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytate-degrading enzymes of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and Bacillus amyloliquefaciens 45 was established using a combination of high-performance ion chromatography analysis and kinetic studies. The data demonstrate that all the Bacillus phytate-degrading enzymes under investigation dephosphorylate myo-inositol hexakisphosphate by sequential removal of phosphate groups via two independent routes; the routes proceed via D-Ins(1,2,4,5,6)P5 to Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3 or D-Ins(2,5,6)P3 and via D-Ins(1,2,4,5,6)P5 to D-Ins(1,2,5,6)P4 to finally D-Ins(1,2,6)P3. The resulting myo-inositol trisphosphate D-Ins(1,2,6)P3 was degraded via D-Ins(2,6)P2 to finally Ins(2)P after prolonged incubation times in combination with increased enzyme concentration.