We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling.