We prepared a cell sheet by using collagenase treatment to digest salmon atelocollagen fibrillar gel (SAC gel) on which human periodontal ligament (HPDL) cells had been cultured. The SAC gel was found to be digested completely within 2 h at a concentration of 50 U of collagenase per mg of collagen. The SAC gel on which HPDL cells were cultured for 10 d was treated with collagenase, resulting in the formation of a detached and shrunken cell sheet. Immunostaining results showed that the cytoskeleton and fibronectin matrix level of the cell sheet were maintained after collagenase treatment. In addition, collagenase treatment had almost no effect on the activities of HPDL cells.