Introduction: The actin cytoskeleton is a network of filamentous actin and associated proteins that have been implicated in the metastasis and progression of ovarian carcinomas. Cytoskeleton regulators include the Rho GTPases RhoA and Cdc42, as well as their upstream activators and downstream effectors. Some regulators have been observed to be overexpressed in ovarian cancers, such as myotonic dystrophy-related Cdc42 binding kinase-α (MRCKα), a kinase acting downstream of Cdc42. Recent data from the Olson Lab revealed that MRCKα interacts with GEF-H1, an activator of RhoA, in ovarian cancer cells. Our work investigates how this association impacts MRCK and GEF-H1 activities in ovarian cancer. Methods: To evaluate MRCKα activity with and without associated GEF-H1, GEF-H1 was knocked down by siRNA and endogenous MRCKα was immunoprecipitated from A2780 ovarian adenocarcinoma cells for in vitro kinase assays. To examine GEF-H1 and RhoA activity, active protein pulldown assays were used to isolate active GEF-H1 or RhoA from OVCAR8 or A2780 ovarian cancer cells treated with MRCKα and MRCKβ inhibitor (BDP9066), as well as with OVCAR8 MRCKα/MRCKβ knockout cells. Results: There was no significant effect on MRCKα activity as measured by myosin light chain phosphorylation, independent of GEF-H1 association. GEF-H1 activity was significantly increased by MRCK inhibition or knockout in OVCAR8 cells. RhoA activity was significantly following MRCK inhibition in OVCAR8 cells, and RhoA activity in MRCK knockout cells is currently being evaluated. Conclusions: MRCK inhibits RhoA signalling via its phosphorylation and inactivation of GEF-H1. These results reveal a potential crosstalk mechanism between these two GTPase mediated pathways, which are speculated to behave in a compensatory manner to promote myosin phosphorylation and cell contractility.