The Drosophila non-claret disjunctional (Ned) kinesin-like protein is required for spindle assembly in oocytes and spindle maintenance in early embryos. Through the action of ATP-dependent microtubule (MT)-binding sites in the head and ATP-independent MT-binding sites in the tail, Ned may bundle and, perhaps, slide MTs relative to each other. Our previous work on the NIT-binding site of the Ned tail domain demonstrated that this site, like the NIT-binding sites of tau, contains basic residues flanked by proline residues and can promote MT assembly and stability. Here, we characterize the interactions of a monomeric Ned tail protein with subtilisin-digested MTs in order to identify sites on the tubulin dimer that interact with the Ned tail. The results provide evidence for four such binding sites per tubulin dimer and support the hypothesis that each binding site consists of a cluster of acidic residues in the C-terminal regions of alpha- and beta-tubulin. (C) 2003 Elsevier Science (USA). All rights reserved.