Osteocyte-like cells were prepared by sequentially treating calvaria from newborn rats with collagenase and chelating agents. On a reconstituted gel of basement membrane components, cells from the third collagenase digest displayed a round shape and expressed the highest level of alkaline phosphatase with minimal osteocalcin deposition into the matrix. On the other hand, cells derived from the interior after EDTA treatment exhibited well-developed dendritic cell processes and expressed essentially no alkaline phosphatase. The latter population also showed quite distinct characteristics such as higher extracellular activities of casein kinase II and ecto-5′-nucleotidase and the extracellular accumulation of a large amount of osteocalcin associated with mineral. These diverse phenotypic and protein expressions as well as the sites from which each population of cells were recovered strongly suggest that we have isolated osteoblastic and osteocytic cells. Bone sialoprotein II was extracellularly phosphorylated by casein kinase II in osteocytic cells but not in osteoblastic cells. We discuss the possibility that differentiation of young osteocytes from osteoblasts may facilitate the biochemical sequence of mineral deposition in the bone matrix.