In the presence of glycerol, the thionucleotide 2-amino-6-mercapto-9-ribofuranosyl purine 5'-triphosphate (S6-GTP) promotes the assembly of 6 S tubulin to form microtubules. Microtubules assembled with this analog show normal stability properties. In the absence of glycerol, few microtubules are formed with S6-GTP; however, many twisted ribbons are evident. Binding of S6-GTP to tubulin from which the associated proteins and exchangeable nucleotide have been removed (Tu(-] produces about a 16% quenching of intrinsic tubulin fluorescence. Fluorescence titrations indicate an apparent Kd for the tubulin S6-GTP complex of about 3 X 10(-8)M. Binding of S6-GTP to Tu(-) also produces a change in its absorption spectrum. The observed difference spectrum has a maximum at 350 nm and negative extrema at 323 and 338 nm. This suggests that the environment of the thioguanine ring is relatively hydrophobic. Competitive displacement studies yield apparent Kd values of about 1.7 X 10(-8)M for GTP and 8.3 X 10(-8) M for GDP. The changes in absorbance and fluorescence which accompany binding provide an excellent approach to the study of the kinetics and mechanisms of nucleotide binding as well as studies of the kinetics of displacement of GTP, GDP, and their analogs.