In bone, mineralization is tightly regulated by osteoblasts and hypertrophic chondrocytes which release matrix vesicles (MVs) and control extracellular ionic conditions and matrix composition. MVs are the initial sites of hydroxyapatite (HA) mineral formation. Despite growing knowledge about their morphology and function, their biogenesis is not well understood. The purpose of this work was to determine the source of MVs in osteoblast lineage, Saos-2 cells, and to check whether MVs originated from microvilli. Microvilli were isolated from the apical plasma membrane of Saos-2 cells. Their morphology, structure, and function were compared with those of MVs. The role of actin network in MV release was investigated by using microfilament perturbing drugs. When examined by electron microscopy MVs and microvillar vesicles were found to exhibit similar morphology with trilaminar membranes and diameters in the same range. Both types of vesicles were able to induce HA formation. Their electrophoretic profiles displayed analogous enrichment in alkaline phosphatase, Na+/K+ ATPase, and annexins A2 and A6. MVs and microvillar vesicles exhibited almost the same lipid composition with a higher content of cholesterol, sphingomyelin, and phosphatidylserine as compared to plasma membrane. Finally, cytochalasin D, which inhibits actin polymerization, was found to stimulate release of MVs. Our findings were consistent with the hypothesis that MVs originated from cell microvilli and that actin filament disassembly was involved in their biogenesis. J. Cell. Biochem. 106: 127–138, 2009. © 2008 Wiley-Liss, Inc.