Once osteoblasts have completed their bone-forming function, they are either entrapped in bone matrix and become osteocytes or remain on the surface as lining cells. Nonetheless, 50-70% of the osteoblasts initially present at the remodeling site cannot be accounted for after enumeration of lining cells and osteocytes. We hypothesized that the missing osteoblasts die by apoptosis and that growth factors and cytokines produced in the bone microenvironment influence this process. We report that murine osteoblastic MC3T3-E1 cells underwent apoptosis following removal of serum, or addition of tumor necrosis factor (TNF), as indicated by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling and DNA fragmentation studies. Transforming growth factor-beta and interleukin-6 (IL-6)-type cytokines had antiapoptotic effects because they were able to counteract the effect of serum starvation or TNF. In addition, anti-Fas antibody stimulated apoptosis of human osteoblastic MG-63 cells and IL-6-type cytokines prevented these changes. The induction of apoptosis in MG-63 cells was associated with an increase in the ratio of the proapoptotic protein bax to the antiapoptotic protein bcl-2, and oncostatin M prevented this change. Examination of undecalcified sections of murine cancellous bone revealed the presence of apoptotic cells, identified as osteoblasts by their proximity to osteoid seams and their juxtaposition to cuboidal osteoblasts. Assuming an osteoblast life span of 300 h and a prevalence of apoptosis of 0.6%, we calculated that the fraction that undergo this process in vivo can indeed account for the missing osteoblasts. These findings establish that osteoblasts undergo apoptosis and strongly suggest that the process can be modulated by growth factors and cytokines produced in the bone microenvironment.