Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.