To ensure accurate chromosome segregation, the spindle checkpoint delays the onset of sister chromatid separation when the spindle is not attached to a kinetochore. Mad2, a component of the checkpoint, targets fission yeast Slp1/budding yeast Cdc20/human p55CDC and prevents it from promoting proteolysis, which is a prerequisite to sister chromatid separation. The protein is localized to unattached kinetochores in higher eukaryotes, and it is thought to be required for activation of the checkpoint as well. In this study, Mad2 and its target Slp1 were visualized in a tractable organism, fission yeast Schizosaccharomyces pombe. When cells were arrested at a prometaphase-like stage, the Mad2-Slp1 complex was stable and the two proteins were colocalized to unattached kinetochores. When the spindle attachment was completed, the complex was no longer detectable and only Mad2 was found associated to the spindle. These results would suggest that unattached kinetochores provide sites for assembly of the Mad2-Slp1 complex. During interphase, Mad2 was localized to the nuclear periphery as well as to the chromatin domain. This localization was abolished in a yeast strain lacking Mad1, a protein that physically interacts with Mad2. Mad1 may anchor Mad2 to the nuclear membrane and regulate its entry into the nucleus.