Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60 C and pH 4.0. The Km value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate ( or phytic acid, Ins P-6) to inositol and inorganic phosphate. The sequence of Ins P-6 hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins ( 1,2,4,5,6) P-5, Ins ( 1,2,5,6) P-4, Ins ( 1,2,6) P-3, Ins ( 1,2) P-2, Ins ( 1 or 2) P-1, and inositol ( notation 3/4/5/6/1 or 2).