We calculated three-dimensional (3D) distribution of water molecules around and inside a complex of a cellulase, Cel44A, with a cellohexaose, based on the 3D-RISM theory. A distinct peak is observed in the 3D-distribution of water at the position within the hydrogen-bond distance from the two residues Glu186 and Glu359 in the enzyme. We identified the water molecule as a substrate of the enzymatic hydrolysis reaction. The finding provides strong support to one of the proposed mechanisms concerning the reaction, that is the retention process. (C) 2008 Published by Elsevier B.V.