Guided tissue regeneration (GTR) is a useful modality in the management of periodontal disease and for bone augmentation around osseointegrated implants. This study evaluated the in vivo use of atelocollagen membrane (AC) on which osteoblastic cells (OBCs) were cultured in vitro, for application as a GTR membranous material. Osteoblastic cells isolated in our laboratory from mouse calvaria formed a thin film on the AC in vitro which was easily manipulated after 21 days in culture. The AC and OBCs complex material (ACOB) was subjected to freezing and thawing and implanted in mouse subcutaneous tissue for the study of histologic events surrounding the implanted ACOB. Histologic findings in the subcutaneous tissue showed calcification on the ACOB at 28 days postimplantation, while no such finding was evident at the control site, where only AC without OBCs were grafted. The present study suggests the possibility of membrane calcification for GTR through ACOB produced by OBCs on an AC in vitro. Keywords: atelocollagen, calcified material, culture, guided tissue regeneration, osteoblastic cell