Limited proteolysis of tubulin by alpha-chymotrypsin cleaved the beta-subunit preferentially at Tyr 281, generating primarily 35 kD and 17 kD fragments which were located in the amino terminal and the carboxy terminal regions, respectively. A small amount of a 19 kD fragment from the C-terminal end was also produced. Alpha-Chymotrypsin-treated tubulin retained the ability to exchange GTP and covalently incorporate nucleotide by direct photoaffinity labeling. SDS-PAGE and autoradiography analysis of the [alpha-32P] GTP-labeled alpha-CT-treated tubulin showed that the 35 kD fragment was almost exclusively labeled, indicating that the exchangeable GTP binding domain resides in the amino terminal region of the beta-subunit.