The goal of this work was to determine the binding properties and location of 4',6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2+/-0.4 microM for the DAPI-tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a kappa2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20+/-2 A and 43+/-2 A, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI.