Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na+, K+-ATPase, protein kinase C, OF alkaline phosphatase to yield inorganic phosphate (Pi), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase I producing inorganic pyrophosphate (PPi), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization. (C) 2009 Elsevier Inc. All rights reserved.