The synthesis of matrix Gla protein (MGP) and bone Gla protein (BGP) have been shown to be mutually exclusive in all osteosarcoma cell lines investigated. In the cell lines that produce the respective proteins, synthesis is stimulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) within the first several hours of hormone treatment. In the present studies we have investigated the effects of longer-term treatment with 1,25(OH)2D3 in the ROS 17/2 cell line, a cell line that synthesizes BGP constitutively but does not synthesize MGP. In agreement with earlier studies, the rate of BGP synthesis increases within 8 hours of hormone treatment, is maximal by 24 hours, and remains at the maximal rate through 48 hours of 1,25(OH)2D3 treatment. The present study is the first to report that the rate of BGP secretion at times beyond 48 hours declines to that of control cultures despite the continued administration of 1,25(OH)2D3, and that MGP synthesis is induced in ROS 17/2 cells by 48 hours of 1,25(OH)2D3 treatment. At this time, MGP mRNA could be detected by northern blot analysis and MGP secretion could be demonstrated by radioimmunoassay of culture medium. Both the level of MGP message per unit total RNA and the rate of MGP secretion into culture medium increased steadily between 2 and 6 days of 1,25(OH)2D3 treatment. The MGP synthesized by the 1,25(OH)2D3-treated ROS 17/2 cells was identical to that found in bone by northern blot analysis of message and by western blot analysis of the media antigen. Half-maximal induction of MGP synthesis was obtained with 0.3 nM 1,25(OH)2D3, a 60-fold higher dosage than was required for the half maximal stimulation of BGP synthesis in these cells. Treatment of ROS 17/2 cells with 24,24-F21,25(OH)2D3 suggests that the observed difference in dose dependence is not due to an increased rate of hormone catabolism.