Tubulin carboxypeptidase is the enzyme that releases the C-terminal tyrosine from alpha-tubulin, converting tyrosine-terminated (Tyr) to detyrosinated (Glu) tubulin. The present study demonstrates that this enzyme is associated with microtubules in living cells. We extracted cultured cells (COS-7) with Triton X-100 under microtubule-stabilizing conditions and found tubulin carboxypeptidase activity in the cytoskeleton fraction. We ruled out, by using several control experiments, the possibility that this result was due to contamination of the isolated cytoskeletons by non-associated proteins contained in the detergent fraction or to an artifact in vitro during the extraction procedure. The associated carboxypeptidase activity showed characteristics similar to those of brain tubulin carboxypeptidase and different from those of pancreatic carboxypeptidase A. In comparison with cultures at confluence, those at low cell density contained small (if any) amounts of carboxypeptidase activity associated with microtubules. In addition, the enzyme was shown to be associated only with cold-labile microtubules. The tubulin carboxypeptidase/microtubule association was also demonstrated in Chinese hamster ovary, NIH 3T3 and PC12 cells. Interestingly, this association was not observed in cultured embryonic brain cells. Our results demonstrate that tubulin carboxypeptidase is indeed associated with microtubules in living cells. Furthermore, the findings that this association occurs with a subset of microtubules and that its magnitude depends on the degree of confluence of the cell culture indicate that it could be part of the mechanism that regulates the tyrosination state of microtubules.