Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1's catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1-C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression. © 2010 Hewitt et al.