Considerable evidence both in vitro and in vivo implicates protein damage by peroxynitrite as a probable mechanism of cell death. Herein, we report that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dose-dependent inhibition of microtubule polymerization. The extent of cysteine oxidation induced by peroxynitrite correlated well with inhibition of microtubule polymerization. Disulfide bonds between the subunits of the tubulin heterodimer were detected by Western blot as a result of peroxynitrite-induced cysteine oxidation. Addition of disulfide reducing agents including dithiothreitol and beta-mercaptoethanol restored a significant portion of the polymerization activity that was lost following peroxynitrite addition. Thus, peroxynitrite-induced disulfide bonds are at least partially responsible for the observed inhibition of polymerization. Sodium bicarbonate protected microtubule protein from the peroxynitrite-induced inhibition of polymerization. Tyrosine nitration of microtubule protein by 1 mM peroxynitrite increased approximately twofold when sodium bicarbonate was present whereas the extent of cysteine oxidation decreased from 7.5 to 6.3 mol cysteine/mol tubulin. These results indicate that cysteine oxidation of tubulin by peroxynitrite, rather than tyrosine nitration, is the primary mechanism of inhibition of microtubule polymerization.