Matrix vesicles (MVs) induce calcification during endochondral bone formation. Experimental methods for structural, compositional, and functional analysis of MVs are reviewed. MV proteins, enzymes, receptors, transporters, regulators, lipids and electrolytes are detailed. MV formation is considered from both structural and biochemical perspectives. Confocal imaging of Ca²⁺ and H⁺ were used to depict how living chondrocytes form MVs. Biochemical studies revealed that coordinated mitochondrial Ca²⁺ and Pi metabolism produce MVs containing a nucleational complex (NC) of amorphous calcium phosphate, phosphatidylserine and annexin A5 – all critical to the mechanism of mineral nucleation. Reconstitution of the NC and modeling with unilamellar vesicles reveal how the NC transforms into octacalcium phosphate, regulated by Mg²⁺, Zn²⁺ and annexin A5. Extravasation of intravesicular mineral is mediated by phospholipases and tissue-nonspecific alkaline phosphatase (TNAP). In the extravesicular matrix, hydroxyapatite crystal propagation is enhanced by cartilage collagens and TNAP, which destroys inhibitory PPi, and by metalloproteases that degrade proteoglycans. Other proteins also modulate mineral formation. Recent findings from single and multiple gene knockouts of TNAP, NPP1, ANK, PHOSPHO1, and Annexin A5 are reviewed.