The carboxypeptidase previously described that releases tyrosine from tubulinyl-tyrosine was obtained from rat brain preparation free of tubulin-tyrosine ligase. The enzyme was purified 24-fold. Its activity was increased by 2 mM MgCl2 or 30 mM KCl. Mercaptoethanol (50 mM), colchicine (0.2 mM) and tyrosine (0.2 mM) showed practically no effect on the release of tyrosine whereas iodoacetate (2 mM), deoxycholate (0.5%), CuCl2 (0.1 mM), ZnCl2 (0.1 mM) and NaCl or KCl (240 mM) had a strong inhibitory effect. The optimal pH of this enzyme was 6.3--7. A preparation containing tubulin-tyrosine ligase free of carboxypeptidase was also obtained. This preparation catalyzed the release of tyrosine from tyrosinated tubulin in the presence of ADP, Mg2+, K+ and Pi and the incorporation of tyrosine into tubulin. For the releasing activity the optimal concentration of MgCl2 was 3--20 mM and of KCl was 10--30 mM. For ADP the maximal act;vity was at 0.3 mM or higher. An important difference between the activities of the carboxypeptidase and the ligase was that the former was active on denatured tubulin whereas the latter was not.