Cellular and molecular events in young (passage 1-3) and aged (passage 25-30) primary mouse aortic smooth muscle cells (MASMC) were investigated. Immunoblot and immunofluorescence analyses indicated that smooth muscle alpha-actin (SM alpha-actin) levels were significantly reduced with increasing in vitro age. Aged MASMC showed an increased proliferative capacity in response to fetal bovine serum (FBS) in comparison with young MASMC. The cell cycle-associated proteins such as cyclin D1, cyclin E, CDK2, and CDK4, and kinase activities associated with CDK2 and CDK4 were increased in aged MASMC. In addition, CDK inhibitor p21 was elevated in aged cell, whereas p27 was decreased. These changes of G1 cell cycle machinery could be explained by the increased proliferative capacity. Matrix metalloproteinase-9 (MMP-9) expression was also increased in response to tumor necrosis factor-alpha (TNF-alpha) in aged MASMC, as evidenced by zymography and immunoblot analysis. Transient transfection assays showed an age-dependent increase in transcription from MMP-9 promoter activity in response to TNF-alpha. In addition, the transcription factors NF-kappaB and AP-1 that are involved in the MMP-9 regulation of aged MASMC in response to TNF-alpha were identified by means of mutation analysis and gel shift assays. These results suggest that the age-associated increase in SMC proliferative capacity, accumulative cell cycle regulators, and MMP-9 expression may play a role in vascular remodeling during in vitro aging.