Countercurrent centrifugal elutriation was used to separate, on the basis of size, isolated growth-plate chondrocytes in chicks. The mean cellular volume, activity of alkaline phosphatase, and synthesis of type-X collagen increased progressively in each of seven successive fractions. Slices of tissues that contained either proliferating or hypertrophic chondrocytes were also removed by manual dissection from the superficial and deep regions of the growth plate. These cells demonstrated differences in size and biochemistry that were similar to those observed in chondrocytes that were separated by elutriation. These differences included increased synthesis of proteoglycan and collagen in the larger chondrocytes. Radiolabeled hypertrophic chondrocytes were mixed with unlabeled resting and proliferating chondrocytes, and then were separated by elutriation. The radioactivity was recovered in fractions that contained the largest cells, confirming that differences in the sizes of the cells can be used to effect a zonal separation by centrifugal elutriation. Clinical relevance: The separation of growth-plate chondrocytes into subpopulations in different stages of maturation has important implications for the study of endochondral ossification. The separated cells maintain a difference in phenotypic expression in short-term culture, providing a model for the study of factors that control the maturation of chondrocytes. Elutriation may prove to be useful in elucidating the cellular events that lead to calcification. Furthermore, this technique of separating cells is potentially applicable to the study of endochondral ossification in cartilaginous tissues that have a less orderly cellular architecture, such as fracture callus, cartilaginous tumors, and heterotopic ossification.