—Vascular calcification is a frequent component of atherosclerosis, yet the pathological mechanisms that regulate its formation are poorly understood. Calcification of the vessel wall may represent a process by which cells that normally exhibit a smooth muscle phenotype differentiate into cells that exhibit an osteoblast-like phenotype. One of the determinants of cellular phenotype is extracellular matrix; thus, we undertook the current study to evaluate the influence of extracellular matrix on calcification of vascular cells in vitro. Cell lines derived from bovine aortic media were divided into 1 of 3 groups: those that did not mineralize, those that mineralized slowly, or those that mineralized rapidly. When slowly mineralizing cells were plated onto matrix produced by rapidly mineralizing cells, the time required for mineralization decreased from 33±3.0 days to 7.8±1.3 days. Matrix produced by rapidly mineralizing cells was found to contain 3 times the amount of collagen I and fibronectin but 70% less collagen IV than nonmineralizing clones. When slowly mineralizing cells were cultured on purified collagen I or fibronectin, mineralized nodule formation, calcium incorporation, von Kossa staining, and alkaline phosphatase activity increased. In contrast, culturing slowly mineralizing cells on purified collagen IV inhibited these mineralization parameters. Furthermore, blocking antibodies to α5 integrins significantly inhibited the fibronectin-mediated increases in alkaline phosphatase activity, indicating that integrin-based signaling may be involved. These data suggest that matrix composition can regulate development of arterial calcification and that a subpopulation of vascular cells preferentially produces positively regulating matrix components.