Tubulin requires GTP for maximal rate and extent of polymerization into microtubules. The localization of the guanine nucleotide in the microtubule was examined by preparing affinity probes that would permit tubulin polymerization prior to their covalent coupling to amino acids in tubulin's exchangeable GTP binding site. Two different hydrolyzable GTP analogues with modified ribose moieties, 3'-p-azidobenzoyl)-GTP and the periodate oxidation product of GTP, 2-(guanylformylmethoxy)-3-(triphospho)propanal, were isolated by thin-layer chromatography and high-voltage electrophoresis and identified by ultraviolet and infrared spectroscopy. The analogues bind to the tubulin molecule and promote polymerization. After tubulin polymerization and isolation of microtubules, the [3H]GTP analogues were covalently coupled to tubulin by NaBH4 reduction or UV irradiation. The microtubules possessed about 1 mol of acid-precipitable 3H-labeled nucleotide/mol of tubulin dimer. Separation of the subunits showed that the nucleotide analogues were associated with both alpha and beta subunits of tubulin in nearly equal amounts. The binding of analogues to both alpha and beta subunits was saturable and competitive with GTP. Cyanogen bromide cleavage of both alpha and beta subunits showed that the 3H-labeled nucleotide was associated with a single molecular weight species of similar size (approximately 10 000) from each subunit. Two-dimensional electrophoresis of chymotryptic peptides from both (alpha and beta) cyanogen bromide fragments showed that the 3H-labeled nucleotide was associated with a peptide of nearly identical migration properties from both subunits. These results suggest that a similar peptide segment of both alpha- and beta-tubulin has the ability to bind GTP. Furthermore, this peptide was localized to the amino-terminal one-third of the tubulin molecule.