Endoreduplication has been suggested to contribute to the development of hypertrophy of smooth muscle cells (SMCs) in hypertension. However, endoreduplication in vascular SMCs and the underlying molecular mechanisms are not clear. We previously demonstrated that treatment of human SMCs with 10 µM 2-methoxyestradiol (2-ME) for 24 h induces accumulation of cells with 4N DNA content and some polyploid/aneuploid cells actively synthesize their DNA, suggesting the occurrence of endoreduplication. In addition, 2-ME treatment up-regulates the expression of cyclin-dependent kinase 2 (Cdk2). The present study was designed to characterize endoreduplication of human SMCs and explore the potential roles of Cdk2 in endoreduplication induced by 2-ME. Treatment with 2-ME (10 µM) for 2 - 4 d not only caused increases in > 4N cells and their re-entry into S phase, but also induced over-duplication of chromosomes. Furthermore, 2-ME increased the kinase activity of Cdk2 and its interaction with cyclin E. Inducible over-expression of dominant-negative Cdk2 in human SMCs inhibited both DNA synthesis of > 4N cells and the accumulation of > 4N cells induced by 2-ME. We conclude that 2-ME induces endoreduplication of human SMCs and Cdk2 plays an important role in endoreduplication in response to 2-ME.