In the fission yeast Schizosaccharomyces pombe, septation and constriction of the actomyosin ring for cell division are positively regulated by the Spg1 GTPase, a member of the Ras superfamily. Spg1 is negatively regulated by Byr4 and Cdc16, which together form a two-component GTPase-activating protein for the Spg1 GTPase. To better understand how Byr4 regulates septation, Byr4 mutants were tested for in vitro functions. This analysis revealed that Byr4 contained one Cdc16-binding site and four Spg1-binding sites (SBS), designated SBS1-SBS4. Although mutants with a single SBS bound Spg1 and inhibited GTP dissociation, the equilibrium binding affinity of these mutants was 28-280-fold weaker than Byr4. Because some Byr4 mutants with multiple SBSs bound Spg1 tighter than the corresponding mutants with a single SBS, multiple SBSs probably interact to cause the high affinity binding of Byr4 to Spg1. A region of Byr4 that bound Spg1, SBS4, and the region that bound Cdc16, Cdc16-binding site, was necessary and sufficient to form Cdc16-dependent Spg1GAP activity that was similar to that of wild-type Byr4 with Cdc16.