We found that excessive extracellular Ca2+ and/or Mg2+ affected the process of matrix mineralization and glycosaminoglycan (GAG) production by cells of the prechondrogenic cell line, ATDC5. Excess Ca2+ induced both matrix mineralization and GAG production in the cells. On the other hand, excess Mg2+ reduced this Ca2+-mediated rise in both mineralization and GAG production in them. Next we measured the mRNA levels of cartilage-associated genes such as calcium-sensing receptor (CaSR), matrix gla protein (MGP), bone gla protein (BGP), and Runt-related transcription factor 2 (Runx2) in ATDC5 cells. Excess Ca2+ increased the MGP, BGP, and CaSR mRNA levels, and excess Mg2+ reduced the Ca2+-induced increase in the MGP mRNA level in the cells. The changes in the MGP mRNA level paralleled those in the MGP protein level. These data show that Ca2+ and Mg2+ regulated the matrix mineralization positively and negatively, respectively, in ATDC5 cells and suggest that excess Mg2+ might inhibit the excess Ca2+-promoted mineralization mediated by MGP induction in chondrocytes. (c) 2006 Elsevier Inc. All rights reserved.