Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately 2-fold increases in c[cyclic]AMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activities were measured in the 2 types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase and general protein synthesis. Although each of these activities was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase were higher in the PT cells. Parathormone stimulated acid phosphatase and hyaluronate synthesis by 100-200% only in the CT cells; it inhibited alkaline phosphatase, citrate decarboxylation and prolyl hydroxylase by 75-90% only in the PT cells. Calcitonin alone had no effect on any of these activities other than cAMP production, but it inhibited the action of parathormone in the CT cells. The sensitivities, time courses of development and magnitudes of these hormonal effects were similar to those observed previously in intact calvaria, indicating that the isolated cell system is a reliable model for the study of bone metabolism. Based on the metabolic responses of the cells, the CT type of populations is probably enriched in osteoclasts and, possibly, osteocytes, and the PT type of population is probably enriched in osteoblasts.