Fluorescence energy transfer experiments were performed in order to measure the spatial separation between the colchine and Ruthenium Red binding sites, the high-affinity bisANS and Ruthenium Red sites, and the allocolchicine and high-affinity bisANS sites on calf brain tubulin. Energy transfer was observed between both colchicine and allocolchicine and Ruthenium Red, resulting in a distance of 40-45 A between these sites on the tubulin molecule. No detectable energy transfer could be observed when allocolchicine was used as fluorescence donor and bisANS as acceptor or when bisANS was used as donor and Ruthenium Red as acceptor. This indicates that the distance of separation between the allocolchicine and bisANS sites is greater than 50 A, while that between the bisANS and Ruthenium Red sites is greater than 72 A. On the basis of these and previous distance measurements (Ward & Timasheff, 1988), two triangles of binding sites have been defined (colchicine-bisANS-E-site and colchicine-bisANS-Ruthenium Red). Since the dihedral angle between them is not known, a schematic model has been drawn with all the sites located in a single plane.(ABSTRACT TRUNCATED AT 250 WORDS)