Hyperproliferation of vascular smooth muscle cells is a hallmark of atherosclerosis and related vascular complications. Microtubules are important for many aspects of mammalian cell responses including growth, migration and signaling. alpha-Tubulin, a component of the microtubule cytoskeleton, is unique amongst cellular proteins in that it undergoes a reversible posttranslational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyr-tubulin). Whereas the reversible detyrosination/tyrosination cycle of alpha-tubulin has been implicated in regulating various aspects of cell biology, the precise function of this posttranslational modification has remained poorly characterized. Herein, we provide evidence suggesting that alpha-tubulin detyrosination is a required event in the proliferation of vascular smooth muscle cells. Proliferation of rat aortic smooth muscle cells in response to serum was temporally associated with the detyrosination of alpha-tubulin, but not acetylation of alpha-tubulin; Glu-tubulin reached maximal levels between 12 and 18h following cell cycle initiation. Inclusion of 3-nitro-l-tyrosine (NO(2)Tyr) in the culture medium resulted in the selective nitrotyrosination of alpha-tubulin, that was paralleled by decreased elaboration of Glu-tubulin, decreased expression of cyclins A and E, decreased association of the microtubule plus-end binding protein EB1, and inhibited cell proliferation. Nitrotyrosination of alpha-tubulin did not induce necrotic or apoptotic death of rat aortic smooth muscle cells, but instead led to cell cycle arrest at the G(1)/S boundary coincident with decreased DNA synthesis. Collectively, these results suggest that the C-terminus of alpha-tubulin and its detyrosination are functionally important as a molecular switch that regulates cell cycle progression in vascular smooth muscle cells.