As part of its overall function as a major regulator of calcium homeostasis, PTH stimulates bone resorption and inhibits osteoblast-mediated biomineralization. To determine the basis for the inhibitory actions of this hormone, we compared the time course of PTH-dependent inhibition of mineralization in MC3T3-E1 osteoblast-like cells with changes in mRNA levels for several extracellular matrix proteins previously associated either with induction or inhibition of mineralization. Mineralizing activity was rapidly lost in PTH-treated cells (∼30% inhibition after 3 h, 50% inhibition at 6 h). Of the proteins examined, changes in matrix γ-carboxyglutamic acid protein were best correlated with PTH-dependent inhibition of mineralization. Matrix γ-carboxyglutamic acid protein mRNA was rapidly induced 3 h after PTH treatment, with a 6-to 8-fold induction seen after 6 h. Local in vivo injection of PTH over the calvaria of mice also induced a 2-fold increase in matrix γ-carboxyglutamic acid protein mRNA. Warfarin, an inhibitor of matrix γ-carboxyglutamic acid protein γ-carboxylation, reversed the effects of PTH on mineralization in MC3T3-E1 cells, whereas vitamin K enhanced PTH activity, as would be expected if a γ-carboxyglutamic acid-containing protein were required for PTH activity. Levels of the other mRNAs examined were not well correlated with the observed changes in mineralization. Osteopontin, an in vitro inhibitor of mineralization, was induced approximately 4-fold 12 h after PTH addition. Bone sialoprotein mRNA, which encodes an extracellular matrix component most frequently associated with mineral induction, was inhibited by 50% after 12 h of PTH treatment. Osteocalcin mRNA, encoding the other known γ-carboxyglutamic acid protein in bone, was also inhibited by PTH, but, again, with a significantly slower time course than was seen for mineral inhibition. Taken together, these results show that the rapid inhibition of osteoblast mineralization induced by in vitro PTH treatment is at least in part explained by induction of matrix γ-carboxyglutamic acid protein.