Chick embryo vertebral chondrocytes (CHECOV cells) grown in agarose gels form spherical colonies containing cells of hypertrophic morphology and a metachromatically staining matrix. Biochemical analysis of these cultures resulted in the following findings. (i) Calcification of CHECOV cultures can be induced by addition of Pi (at least 1.9 mM) or beta-glycerol phosphate (BGP). (ii) Alkaline phosphatase activity reaches a maximal value at the time when mineral deposition is initiated. (iii) Added

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... BGP is converted to Pi; maximal production of Pi occurs at the time of maximal alkaline phosphatase activity. (iv) BGP-supplemented cultures produce a degree of calcification that corresponds to the amount of BGP conversion to Pi. It can be concluded that Pi is rate-limiting for the calcification of chondrocyte cultures. BGP promotes calcification of these cultures by acting as a substrate for the alkaline phosphatase-mediated production of inorganic phosphate.