A rapid and sensitive assay for [3H]GTP binding activity of tubulin has been developed. This assay method is based on the quantitative retention of [3H]GTP-tubulin complex on a nitrocellulose membrane filter. It was also found that bovine brain tubulin is markedly stabilized by glycerol and GTP against denaturation. A large-scale purification of bovine brain tubulin was achieved using the new assay procedure and by the inclusion of glycerol and GTP in a buffer solution used for column chromatography. The purified tubulin could be stored at −80° in the presence of glycerol and GTP for at least a year without any appreciable loss of [3H]GTP- and [3H]colchicine binding activities. The interaction of tubulin with guanine nucleotides was also studied using the nitrocellulose membrane filter procedure. It was found that the binding of [3H]GTP to tubulin with an empty exchangeable site proceeded promptly within 5 sec while the exchange of [3H]GTP with a GTP-tubulin complex in which the exchangeable site had been occupied with unlabeled GTP occurred more slowly. The dissociation constants for GTP and GDP at the exchangeable site of tubulin were determined as 0.5×10−5 M and 1.9×10−5 M, respectively. 5′-Guanylylimidodiphosphate could interact, although less strongly, with tubulin at this site, whereas the interaction of other nucleoside triphosphates including ATP, CTP, UTP, and 5′-guanylyl methylenedi-phosphonate was very weak, if it occured at all. The presence of Mg2+ and a free sulfhydryl group was found to be essential for binding of [3H]GTP to tubulin. Ca2+ was found to replace Mg2+ in this binding reaction.