The mechanical separation of rat skeletal tissue into viable populations of periosteal cells, osteoblasts, osteocytes, and marrow cells has been undertaken. Periosteal cells and osteocytes were obtained as tissue preparations, while osteoblasts and marrow cells were isolated as individual cells. The cell populations were identified during the preparative procedure by their histological and histochemical appearance. Viability of these preparations was demonstrated by their metabolic activity during short-term incubations and their histological appearance and vital staining before and after such incubations. The cell populations produced carbon dioxide and lactate, consumed oxygen, and incorporated uridine into RNA. Comparison of uridine incorporation into RNA of cells from control and parathyroid hormone treated rats was compatible with a differential effect of parathyroid hormone on the cell populations. These findings indicate that the technique described can be utilized to produce viable bone cells of different types, and may offer a new means for characterizing the role of each cell type in the metabolism of skeletal tissue.