The in vivo metabolism of 32P orthophosphate into phospholipids of chondroxyte, matrix vesicle, and membrane fractions of chicken epiphyseal cartilage has been studied. Incorporation of radioactive phosphate into the total phospholipid fraction of matrix vesicles was rapid, the labeling of phosphatidylcholine and lysophosphatidylserine being even more rapid in matrix vesicles than in chondrocytes. These findings indicate that matrix vesicles are formed by a rapid, metabolically active process, and are not remnants of dead cells, as had previously been postulated by some workers. The rate of incorporation of 32P orthophosphate into phosphatidylserine and sphingomyelin of matrix vesicles was significantly slower than that of phosphatidylcholine and certain other vesicle phospholipids. These findings are paradoxical because, compared with chondrocytes, matrix vesicles were enriched in phosphatidylserine and sphingomyelin and depleted in phosphatidylcholine. These results indicate that in vesicle formation the rates of degradation of the various phospholipid classes must be markedly different: phosphatidylcholine must be degraded much more rapidly than either phosphatidylserine or sphingomyelin. Support for this comes from previous data which revealed that substantial phospholipase activity is present in epiphyseal cartilage, especially in the zone of hypertrophy where matrix vesicle formation appears to be particularly active.