The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ-tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time-lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co-localize at the spindle pole body. SAC activity is, thus, spatially regulated in A.nidulans. Likewise, Cdc20, an activator of the anaphase-promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ-tubulin mutation mipAD159 causes a nuclear-specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2.