The protein G18 (also known as AGS4 or GPSM3) contains three conserved GoLoco/GPR domains in its central and C-terminal regions that bind to inactive Gαi, whereas the N-terminal region has not been previously characterized. We investigated whether this domain might itself regulate G protein activity by assessing the abilities of G18 and mutants thereof to modulate the nucleotide binding and hydrolytic properties of Gαi1 and Gαo. Surprisingly, in the presence of fluoroaluminate (AlF4-) both G proteins bound strongly to full-length G18 (G18wt) and to its isolated N-terminal domain (G18ΔC) but not to its GoLoco region (ΔNG18). Thus, it appears that its N-terminal domain promotes G18 binding to fluoroaluminate-activated Gαi/o. Neither G18wt nor any G18 mutant affected the GTPase activity of Gαi1 or Gαo. In contrast, complex effects were noted with respect to nucleotide binding. As inferred by the binding of [ 35S]GTPγS (guanosine 5′-O-[γ-thio]triphosphate) to Gαi1, the isolated GoLoco region as expected acted as a guanine nucleotide dissociation inhibitor, whereas the N-terminal region exhibited a previously unknown guanine nucleotide exchange factor effect on this G protein. On the other hand, the N terminus inhibited [35S]GTPγS binding to Gαo, albeit to a lesser extent than the effect of the GoLoco region on Gαi1. Taken together, our results identify the N-terminal region of G18 as a novel G protein-interacting domain that may have distinct regulatory effects within the Gi/o subfamily, and thus, it could potentially play a role in differentiating signals between these related G proteins.