During mitosis, replicated chromosomes are equally distributed among two daughter cells by means of a multi-component machine called the mitotic spindle. Spindle formation and function has been shown to involve numerous microtubule associated proteins and molecular motor proteins, including kinesins and dynein. One such kinesin, the plus-end directed, homotetrameric, Eg5, is involved in centrosome separation during spindle formation. In vitro, Eg5 crosslinks parallel and antiparallel microtubules, and localizes to spindle poles and microtubules in vivo. To further understand the function of Eg5 in mammalian cells, we determined its distribution and dynamics throughout mitosis using novel cloning techniques, fluorescence recovery after photobleaching, and total internal reflection fluorescence microscopy. Eg5-GFP was expressed from a mouse bacterial artificial chromosome to ensure the transgene’s expression was at or near endogenous levels. Our results confirm that Eg5 colocalizes with spindle, but not astral, microtubules and is enhanced at the spindle poles during prometaphase and metaphase. In early anaphase, Eg5 is localized near the poles transitioning to interzone microtubules with the exception of a 1 µm gap during late anaphase. Fluorescence recovery after photobleaching shows that Eg5 is rapidly turning over throughout mitosis with a recovery half time less than 10 s and extent of recovery greater than 85%. TIRF microscopy revealed a population of Eg5 that transiently binds to microtubules with a residency time of less than 6 seconds for all stages of mitosis. Eg5 remained stationary while bound to microtubules with no apparent directional motion. Treatment of cells expressing mEg5-GFP-LAP with the Eg5 inhibitor, STLC, caused Eg5 to no longer bind to microtubules and remain diffuse within the cell. TIRF microscopy also revealed Eg5-decorated tracks during interphase, which were abolished by treatment with STLC or Nocodazole suggesting that Eg5 is present on microtubules in interphase. Taken together, fluorescence recovery after photobleaching and TIRF microscopy reveal that Eg5 is highly dynamic in the mammalian spindle throughout mitosis.