We examined the distribution of the dynein-associated protein NudE in Drosophila larval brain neuroblasts and spermatocytes, and analyzed the phenotypic consequences of a nudE null mutation. NudE can associate with kinetochores, spindles and the nuclear envelope. In nudE mutant brain mitotic cells, centrosomes are often detached from the poles. Moreover, the centrosomes of mutant primary spermatocytes do not migrate from the cell cortex to the nuclear envelope, establishing a new role for NudE. In mutant neuroblasts, chromosomes fail to congress to a tight metaphase plate, and cell division arrests because of spindle assembly checkpoint (SAC) activation. The targeting of NudE to mitotic kinetochores requires the dynein-interacting protein Lis1, and surprisingly Cenp-meta, a Drosophila CENP-E homolog. NudE is non-essential for the targeting of all mitotic kinetochore components tested. However, in the absence of NudE, the 'shedding' of proteins off the kinetochore is abrogated and the SAC cannot be turned off, implying that NudE regulates dynein function at the kinetochore.