ADP-ribosylation factors (Arfs), key regulators of intracellular membrane traffic, are known to exert multiple roles in vesicular transport. We previously isolated eight temperature-sensitive (ts) mutants of the yeast ARF1 gene, which showed allele-specific defects in protein transport, and classified them into three groups of intragenic complementation. In this study, we show that the overexpression of Glo3p, one of the GTPase-activating proteins of Arf1p (ArfGAP), suppresses the ts growth of a particular group of the arf1 mutants (arf1-16 and arf1-17). Other ArfGAPs do not show such a suppression activity. All these ArfGAPs show sequence similarity in the ArfGAP catalytic domain, but are divergent in the rest of molecules. By domain swapping analysis of Glo3p and another ArfGAP, Gcs1p, we have shown that the non-catalytic C-terminal region of Glo3p is required for the suppression of the growth defect in the arf1 ts mutants. Interestingly, Glo3p and its homologues from other eukaryotes harbor a well-conserved repeated ISSxxxFG sequence near the C-terminus, which is not found in Gcs1p and its homologues. We name this region the Glo3 motif and present evidence that the motif is required for the function of Glo3p in vivo.