Cytoplasmic dynein is a minus-end directed microtubule-based motor protein that is responsible for the transport and positioning of diverse cellular structures within eukaryotic cells, including membranous organelles and chromosomes during mitosis. Previous work identified a phosphorylation site in one of the dynein subunits (intermediate chains (ICs), which regulates binding to the putative receptor complex dynactin during interphase (Vaughan et al., 2001). To identify dynein kinases and to further investigate dynein cargo-targeting mechanisms, we examined mitotic dynein and determined that the mitotic ICs are phosphorylated by gel-shift and 2D gel analysis. ICs were subjected to MS/MS analysis revealing two novel phosphorylation sites. T89 falls within the binding domain for dynactin, and site-directed mutants designed to mimic a phosphorylated state reduce dynactin binding significantly. T89 fits the consensus for p38MAPK, and phosphorylation by p38MAPK is specific for this site in vitro. Both dynein and p38 have been independently implicated in kinetochore checkpoint roles during the spindle assembly checkpoint. Inhibition of p38MAPK results in redistribution of dynein from kinetochores to spindle poles and induces dispersed lagging chromosomes. Phospho-specific antibodies reveal that ICs phosphorylated at T89 bind to pro-metaphase kinetochores and are absent from kinetochores that have achieved microtubule attachment and chromosome alignment on the metaphase plate. In contrast, ICs phosphorylated at Y130 localize to mitotic spindle poles and reside there from early prophase until late anaphase. Y130 falls within a domain implicated in LC8 (a dynein light chain) binding and fits the consensus for phosphorylation by c-abl kinase. C-abl phosphorylates wild-type, but not Y130F mutant ICs in vitro. This phosphorylation was blocked by treatment with Gleevec (a c-abl inhibitor) and Gleevec treatment of live cells also blocked phosphorylation at Y130, suggesting that c-abl is a mitotic dynein kinase. Binding assays to test the impact of Y130 phosphorylation demonstrated that ICs phosphorylated at Y130 do not bind LC8. These results suggest that phosphorylation of dynein ICs modulates subunit composition during mitosis and plays a role targeting dynein ICs to specific functional mitotic sites.