We have measured the activity of the pentose shunt pathway in the chick growth cartilage. Measurement of D-[1-¹⁴C] glucose and D-[6-¹⁴C] glucose metabolism by chondrocytes indicated that pentose phosphate shunt activity was low. However, when the cells were stimulated with phenazine methosulfate (PMS) and t-butyl hydroperoxide, a significant elevation in shunt activity was observed. This activity was further increased by dithiothreitol. Enzymatic and substrate requirements of the shunt pathway were examined and related to morphology of the tissue. It was found that as chondrocytes mature, there is increased glucose-6-phosphate dehydrogenase activity, and decreased quantities of glucose-6-phosphate and NADPH. While these investigations indicated that shunt activity was maximum in hypertrophic cartilage, the results of cytochemical studies suggested that the activity was greatest in those cells that were most removed from the O₂ supply. Experiments were performed to examine O₂ requirements of chondrocytes in relationship to the pentose phosphate shunt. First, using a phosphorescence quenching technique, total O₂ uptake by these cells was found to be constant over a large part of the physiological range of O₂ tensions. Over the same range, when stimulated by PMS, O₂ uptake by CN⁻ treated cells was increased. In the 1–5 μM O₂ range, non-mitochondrial O₂ consumption decreased more slowly than total respiration. Finally, the observation that NADPH directly stimulated chondrocyte O₂ consumption suggest that cartilage cells may be able to form O₂ metabolites.