Using a recently developed technique for measuring proteoglycan by x-ray microprobe analysis, we examined six-week-old rat growth plates. In the proliferating zone, the proteoglycan concentration was found to be relatively low in the transverse septa, but somewhat higher in the longitudinal septa between the proliferating chondrocytes. In the lower hypertropic zone, in the region of the degenerating hypertrophic chondrocytes, the immediate pericellular area had a very high concentration of proteoglycan, as was obtained. These results indicate that either proteoglycan concentrations fall to very low levels in fully mineralized cartilage, or that the mineralization of cartilage interferes in some way with the measurement of microprobe signals from proteoglycans. We speculate that high levels of proteoglycan are necessary for mineralization in the epiphysis of the rat. Clinical Relevance: The spot-to-spot analysis of proteoglycan by microprobe demonstrates the usefulness of this tool in analyzing minute volumes of cartilage for proteoglycan. It further demonstrates that mineralization of growth-plate cartilage is associated with changing patterns of concentration of proteoglycan. These normal patterns may constitute a basis for comparison in similarly evaluating concentrations of proteoglycan in diseases of growth disturbance.