We have previously reported that anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. In this study, we show that tubulin is present in mitochondria isolated from different human cancerous and non-cancerous cell lines. The absence of polymerized microtubules and cytosolic proteins was checked to ensure that this tubulin is an inherent component of the mitochondria. In addition, a salt wash did not release the tubulin from the mitochondria. By using electron microscopy, we then showed that tubulin is localized in the mitochondrial membranes. As compared with cellular tubulin, mitochondrial tubulin is enriched in acetylated and tyrosinated alpha-tubulin and is also enriched in the class III beta-tubulin isotype but contains very little of the class IV beta-tubulin isotype. The mitochondrial tubulin is likely to be organized in alpha/beta dimers and represents 2.2 +/- 0.5% of total cellular tubulin. Lastly, we showed by immunoprecipitation experiments that the mitochondrial tubulin is specifically associated with the voltage-dependent anion channel, the main component of the permeability transition pore. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with the permeability transition pore.