Matrix Gla protein (MGP) is an inhibitor of vascular calcification but its mechanism of action and pathogenic role are unclear. This was examined in cultured rat aortas and in a model of vascular calcification in rats with renal failure. Both carboxylated (GlaMGP) and uncarboxylated (GluMGP) forms were present in aorta and disappeared during culture with warfarin. MGP was also released into the medium and removed by ultracentrifugation, and similarly affected by warfarin. In a high-phosphate medium, warfarin increased aortic calcification but only in the absence of pyrophosphate, another endogenous inhibitor of vascular calcification. Although GlaMGP binds and inactivates bone morphogenic protein (BMP)-2, a proposed mediator of vascular calcification through up-regulation of the osteogenic transcription factor runx2, neither warfarin, BMP-2, nor the BMP-2 antagonist noggin altered runx2 mRNA content in aortas, and noggin did not prevent warfarin-induced calcification. Aortic content of MGP mRNA was increased 5-fold in renal failure but did not differ between calcified and noncalcified aortas. Immunoblots showed increased GlaMGP in noncalcified (5-fold) and calcified (20-fold) aortas from rats with renal failure, with similar increases in GluMGP. We conclude that rat aortic smooth muscle produces both GlaMGP and GluMGP in tissue-bound and soluble, presumably vesicular, forms. MGP inhibits calcification independent of BMP-2-driven osteogenesis and only in the absence of pyrophosphate, consistent with direct inhibition of hydroxyapatite formation. Synthesis of MGP is increased in renal failure and deficiency of GlaMGP is not a primary cause of medial calcification in this condition.