The formation of isoaspartyl sites during aging of rat tubulin in vitro and in vivo has been studied. When incubated in vitro at pH 7.4, 37 degrees C, purified rat brain tubulin accumulated isoaspartyl sites at a rate > or = 2.4 isoaspartyl sites per 100 tubulin subunits (50 kDa) per day for 30 days. Isoaspartate levels were estimated by the transfer of radiolabeled methyl groups from S-adenosyl-L-[methyl-3H]-methionine in a reaction catalyzed by protein-L-isoaspartyl methyltransferase. isoaspartate formation occurred in parallel with, but was not dependent upon, extensive cross-linking of tubulin via formation of intermolecular disulfide bonds. When rat PC12 cells were incubated for 24 or 72 h in the presence of adenosine dialdehyde, a potent methyltransferase inhibitor, a substantial and consistent increase in the isoaspartate content of tubulin was observed. This suggests that tubulin constantly undergoes isoaspartate formation in vivo, but that the levels are normally kept low by methylation-dependent repair. These findings support the hypothesis that protein-isoaspartyl methyltransferase plays a key role in countering spontaneous damage reactions to proteins associated with cell aging. These results also suggest that tubulin is an important target for protein-isoaspartyl methyltransferase in vivo.