Tumor necrosis factor alpha (TNF-alpha) induces apoptosis in a number of cell types and plays an essential role in bone remodeling, both stimulating the proliferation of osteoblasts and activating osteoclasts. During endochondral ossification, apoptosis of chondrocytes occurs concurrently with new bone formation and the resorption and replacement of mineralized cartilage with woven bone. In the present study, the role of TNF-alpha in promoting chondrocyte apoptosis was examined. Chondrocyte cell populations, enriched in either hypertrophic or non-hypertrophic cells, were isolated from the cephalic and caudal portions of 17-day chick embryo sterna, respectively, and treated in vitro with 0.1-10 nM recombinant human TNF-alpha. As a positive control, apoptosis was also induced by Fas receptor antibody binding. Dye exclusion assays of the live/dead ratios of cells showed that TNF-alpha caused a dose-dependent 1.5- and 2.0-fold increase in the number of dead cells in both hypertrophic and non-hypertrophic chondrocytes. Induction of apoptosis was independently assayed by measurement of interleukin-1beta-converting enzyme (ICE) activity, and analyzed by a semi-quantitative determination of DNA fragmentation. When compared to untreated cells, these analyses also showed dose-dependent increases in TNF-alpha induced apoptosis in both chondrocyte populations, with increases in the levels of ICE activity for all doses of TNF-alpha (from approximately 5 to approximately 20 fold). Osteoblasts, however, were not affected by treatment with TNF-alpha or by Fas antibody/protein G induction. Immunostaining of chondrocytes for Fas receptor and caspase-2 protein expression showed that most of the chondrocytes expressed these two markers of apoptosis after treatment with TNF-alpha. Although cell killing and ICE induction were higher in the more hypertrophic cells, TNF-alpha induced apoptosis in both hypertrophic and non-hypertrophic chondrocyte populations. These results demonstrate that apoptosis may be induced in both hypertrophic and non-hypertrophic chondrocytes through both Fas and TNF-alpha receptor mediated signaling, and suggest that chondrocytes are more sensitive to apoptotic effects of TNF-alpha within the skeletal lineage than are osteoblasts.