Cytohesins are guanine-nucleotide exchange factors (GEF) for the Arf family of GTPases. One member of the Arf family, ARF6, plays an active role in the intracellular trafficking of G protein-coupled receptors. We have previously reported that Galphaq signaling leads to the activation of ARF6, possibly through a direct interaction with cytohesin-2/ARNO. Here, we report that Galphaq can directly interact with cytohesin-1, another Arf-GEF of the ARNO/cytohesin family. Cytohesin-1 preferentially associated with a constitutively active mutant of Galphaq (Galphaq-Q209L) compared to wild-type Galphaq in HEK293 cells. Stimulation of TPbeta, a Galphaq-coupled receptor, to activate Galphaq resulted in the promotion of a protein complex between Galphaq and cytohesin-1. Confocal immunofluorescence microscopy revealed that wild-type Galphaq and cytohesin-1 co-localized in intracellular compartments and at or near the plasma membrane. In contrast, expression of Galphaq-Q209L induced a drastic increase in the localization of cytohesin-1 at the plasma membrane. Expression of a dominant-negative mutant of cytohesin-1 reduced by 40% the agonist-induced internalization of TPbeta, a process that we previously demonstrated to be dependent on Galphaq-mediated signaling and Arf6 activation. Using deletion mutants, we show that cytohesin-1 interacts with Galphaq through its N-terminal coiled-coil domain. Cytohesin-1 and cytohesin-2/ARNO mutants lacking the coiled-coil domain were unable to relay Galphaq-mediated activation of Arf6. This is the first report of an interaction between the coiled-coil domain of the cytohesin/ARNO family of Arf-GEFs and a member of the heterotrimeric G proteins.