In budding yeast, exit from mitosis is achieved by inactivation of Cdc28/Clb2 activity. Although it is not clear at present how mitotic exit is triggered, a growing body of evidence suggests that the Tem1 GTPase plays a critical role in mediating this pathway and that Bfa1 and Bub2 constitute a two-component GTPase-activating protein to negatively regulate Tem1. Here, we have demonstrated that introduction of bfa1 Delta suppresses the growth defects associated with the cdc5-1 mutation significantly better than that of bub2 Delta, suggesting that Bfa1 may have a previously uncharacterized role in this pathway. Overexpression of BFA1 efficiently arrested the cell cycle at postanaphase even in the absence of BUB2, whereas overexpression of BUB2 weakly induced mitotic arrest only in the presence of BFA1. Coimmunoprecipitation and in vitro binding studies indicate that Bfa1 binds strongly to Tem1 independently of Bub2. Provision of GDP+AlF(4)(-), which mimics the GTPase transition state, enhanced the Bub2-Tem1 interaction both in vitro and in vivo. Interestingly, introduction of bfa1 Delta, but not bub2 Delta, greatly increased the interaction between Tem1 and Cdc15, a step that is thought to be critical for activating the mitotic exit network. Our data suggest that, in addition to its role as a putative, two-component GTPase-activating protein with Bub2, Bfa1 also can play a role in the regulation of mitotic exit by directly inhibiting the interaction between Tem1 and Cdc15 even in the absence of Bub2.