Microtubule-nucleating ability of the centrosome in the isolated mitotic apparatus was investigated under the condition that the centrosomal fragment was preincubated with guanine nucleotides or its derivatives. GTP and non-hydrolyzable GTP analogues such as GTP-γS or GMP-PNP consistently supported high activity of the centrosome to nucleate microtubules. On the other hand, GDP substantially suppressed the microtubule-nucleating activity of the centrosomes. Evidence has been presented that this is not simply due to the inhibitory effect of GDP on microtubule assembly. This result is consistent with the idea that GTP binding proteins such as 51 kDa protein and γ-tubulin localized to the pericentriolar materials in the centrosome are competent for the nucleation of microtubules when it is saturated with GTP.