The potential of certain types of cancer cells to spread to secondary sites within the body, or metastasize, as well as other critical cellular events, has previously been shown to be regulated by the monomeric GTPase ADP-ribosylation factor (ARF) 6 [1]. Being a small GTPase, ARF6 cycles between a GDP- and a GTP-bound conformation, and this cycling is required for ARF6-dependent signalling events. ARF6 has no intrinsic GDP/GTP exchange activity or GTPase activity, and so its GDP/GTP cycle is regulated entirely by its interaction with additional regulatory proteins. One such group of these regulatory proteins is a family of phosphoinositide (PI) binding proteins termed cytohesins. Cytohesins function as guanine nucleotide exchange factors (GEFs) for ARF GTPases [2], thereby increasing the amount of ARF6 that is present in its GTP-bound form. We propose that Exchange Factor for ARF6 (EFA6)D, initially identified as a marker for hepatocellular carcinoma cells [3], is a novel member of the cytohesin family, which we have termed cytohesin 7. The expression of cytohesin 7 has been shown to be upregulated in colon cancer and downregulated in prostate cancer [3,4]. In this study we have functionally characterised cytohesin 7 in order to understand how the activation of ARF6 by cytohesin 7 alters the phenotype of the cancer cells. Through localisation studies in PC12 and HeLa cells of GFP-tagged proteins, we have identified that cytohesin 7 is localised to the plasma membrane, and that this localisation is dependent on both its coiled coil (CC) domain and its PI-binding PH domain. Through an in vivo GST effector pull down assay, we have shown that cytohesin 7 functions as a GEF for ARF6, but not ARF1, and that this function is dependent on the presence of a functional Sec7 catalytic domain. Furthermore, exogenous expression of cytohesin 7 in HeLa cells promotes cytoskeleton reorganisation possibly through ARF6 activation. Using artificially membrane-targeted deletion constructs of cytohesin 7, we have also shown that the GEF function of cytohesin 7 towards ARF6 is dependent on its localisation to the plasma membrane. Future studies will be directed towards understanding the role of cytohesin 7 in ARF6 associated cellular functions in normal and diseased states.