Kinesins are molecular motors that transport cargo along microtubules (MTs). To move forward the motor must attach to the MT in a defined orientation and detach from it in a process that is driven by ATP hydrolysis. The knowledge of the motor-MT interface is essential for a detailed understanding of how kinesins move along MTs and how they are related to other molecular motors such as myosins or dyneins. We have used the marine natural product adociasulfate-2 (AS-2), previously identified as a MT-competitive inhibitor of conventional kinesin, to infer the secondary structure elements forming the MT interface of two human mitotic kinesins, namely, CENP-E and Eg5. AS-2 inhibits both basal and MT-stimulated ATPase activities of CENP-E (IC50 of 8.6 and 1.3 microM, respectively) and Eg5 (IC50 of 3.5 and 5.3 microM, respectively) and is a MT-competitive inhibitor of CENP-E with a Ki of 0.35 microM. Binding of AS-2 to CENP-E also stimulates the ADP release from the nucleotide-binding pocket. AS-2 is a nonspecific kinesin inhibitor targeting several superfamily members including KHC, MPP1, MKLP1, RabK6, KIFC1, KIFC3, CENP-E, and Eg5. By measuring hydrogen/deuterium exchange with mass spectrometry we have shown that the formation of the CENP-E/AS-2 complex decreases the solvent accessibility of three neighboring peptides on the same face of CENP-E. We deduce that this is the site of MT attachment and conclude that loop L11, helix alpha4, loop L12, helix alpha5, loop L8, and strand beta5 constitute the main MT interface of the CENP-E motor domain. Similarly for Eg5/AS-2, a region of increased solvent accessibility locates the MT interface of Eg5.