The spindle checkpoint prevents anaphase from occurring until all chromosomes have attached properly to the mitotic spindle. The checkpoint components Mad1 and Mad2 associate with unattached kinetochores and are probably involved in triggering the checkpoint. We now demonstrate that in Xenopus egg extracts Mad1 and Mad2 form a stable complex, whereas a fraction of Mad2 molecules is not bound to Mad1. The checkpoint establishment and maintenance are lost upon titrating out free Mad2 with an excess of Mad1 or a truncated Mad1 (amino acids 326-718, Mad1C) that contains the Mad2-binding region. Mad1N (amino acids 1-445) that binds kinetochores, but not Mad2, reduces Mad1 and Mad2 at kinetochores and abolishes checkpoint maintenance. Furthermore, the association between Mad2 and Cdc20, the activator for the anaphase-promoting complex, is enhanced under checkpoint-active condition compared with that at metaphase. Immunodepletion analysis shows that the Mad1-free Mad2 protein is unable to bind Cdc20, consistent with the model that kinetochore localization of Mad2 facilitates the formation of Mad2-Cdc20 complex. This study demonstrates that the ratio between Mad1 and Mad2 is critical for maintaining a pool of Mad1-free Mad2 that is necessary for the spindle checkpoint. We propose that Mad2 may become activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1-free Mad2.