ACK is a non-receptor tyrosine kinase and an effector protein for Cdc42. ACK has been implicated in carcinogenesis events especially in promoting cell migration and invasion, indicating a need for a fuller understanding of the cellular roles of ACK both in normal and pathological conditions. A previous yeast-2-hybrid screen in the lab identified RhoGDI-3 as an interacting partner for ACK. RhoGDI-3 is a member of RhoGDI family of proteins, which are negative regulators that maintains Rho-family GTPases in the inactive GDP-bound state and sequesters them in the cytosol. RhoGDIs have also been found to become deregulated in cancer and it is possible therefore that they play role in ACK-driven cancer progression. In this work, it is shown that ACK binds but does not phosphorylate the RhoGDIs in cells or in vitro. ACK was shown to shuttle between the cytoplasm and the nucleus. RhoGDI-1 and -2 have been found only in the cytoplasm, while RhoGDI-3 is localized to both cytoplasm and the nucleus, under the conditions tested. The interaction between RhoGDI-3 and ACK occurs predominantly in the nucleus and RhoGDI-3 levels were shown to decrease following co-expression with ACK, especially in the nucleus. Data generated here shows that ACK-mediates RhoGDI-3 proteasomal degradation potentially by regulating RhoGDI-3 ubiquitination. In order to determine the cellular effects of the interaction between ACK and the RhoGDIs, all possible Rho-family GTPases that interact with the RhoGDIs were determined in a systematic study. RhoGDI-1 and 2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity interacting with several novel interacting partners within the Rho, Rac and Cdc42 subfamilies. Unexpectedly RhoGDI-3 was found to form complexes with the atypical Rho GTPases such as RhoD, Wrch2, Rnd2, Miro2 and RhoH which are not regulated by standard GDP/GTP cycling. The GTP levels of these target proteins were found to decrease following co-expression with RhoGDI-3, confirming its role as a negative regulator of these Rho GTPases. ACK was shown to regulate the activation of these target proteins, including RhoA, RhoB, Rac1 and RhoH, which are known to be involved in cell proliferation and migration. Aberrant activation of these target protein is frequently observed in cancer, suggesting a role for RhoGDI to drive ACK oncogenicity.