The correlation between the time courses of pure tubulin assembly and accompanying guanosine 5'-triphosphate (GTP) hydrolysis has been studied at different tubulin concentrations in the range where the rate of assembly varies with a strong cooperativity. One GTP molecule was found hydrolyzed per molecule of tubulin dimer incorporated in the microtubule. This hydrolysis was not strictly coupled to polymerization and occurred in a subsequent step. Consequently, in the first stages of assembly, tubulin-GtP complex is the transient major constituent of microtubules. Kinetic data of GTP hydrolysis have been treated within a model of two consecutive first-order reactions: [tubulin-GTP]free k1 leads to [tubulin-GTP]MT k2 leads to [tubulin-GDP]MT + [Pi] GTP hydrolysis proceeded at an intrinsic rate k2 = 0.25 min-1 independent of tubulin concentration. Simultaneous measurements of polymerization, GTPase activity, and incorporation of [3H]GTP followed by unlabeled GTP chase indicated that before its hydrolysis GTP bound to microtubules was exchangeable while after hydrolysis GDP remained locked in the E site. The possibility is discussed that after assembly tubulin undergoes a conformation change which could trigger GTP hydrolysis and sequestration of GDP.