To isolate specific markers of both differentiated and proliferating vascular smooth muscle cells (VSMCs), we used the technique of differential cDNA screening using RNA from cultured rat aortic VSMCs. The tissue specificity of expression of all of the cDNAs isolated was determined by Northern analysis. We isolated seven distinct cDNAs that were more strongly expressed in freshly dispersed, differentiated, aortic VSMCs compared with dedifferentiated late-passage cells. These were the cDNAs for tropoelastin, a matrix protein; alpha-smooth muscle (SM) actin, gamma-SM actin, calponin, and phospholamban, which are all proteins associated with the contractile function of differentiated VSMCs; SM22 alpha, a smooth muscle-specific protein of unknown function, and CHIP28, a putative membrane channel protein that is not highly expressed in other SM tissues and may therefore be a new VSMC marker. Two cDNAs that were expressed preferentially in late-passage dedifferentiated VSMCs were also isolated. These were the cDNAs for osteopontin and matrix Gla protein (MGP). Like CHIP28, MGP was strongly expressed in aortic VSMCs but not in other types of tissues containing SM cells, suggesting that both have specific functions in vascular tissue. Osteopontin and MGP have both previously been isolated from developing bone. Their expression in proliferating VSMCs suggests that they may be involved in regulating the calcification that commonly occurs in vascular lesions. The set of cDNAs obtained extends the range of DNA probes that are available for identifying VSMCs and characterizing their phenotype in vivo by in situ hybridization. Therefore, they should aid in the analysis of gene expression during the development of vessel lesions.