Well-controlled alkylthiol surface chemistries were used to monitor and modulate the activation state of RhoA in attaching cells. Activation states were determined indirectly by fractionating cell lysates into membrane and cytosolic fractions by ultracentrifugation. Western blots were then performed, showing RhoA localization to be surface chemistry-dependent. RhoGDI levels and its intracellular localization were also shown to be surface-chemistry dependent. Cells cultured on –CH3 terminated SAMs, which normally exhibit a low-growth phenotype, were transfected with a constitutively active mutant form of RhoA. Subsequent cell morphological changes were observed on SAM surfaces by fluorescence microscopy. Results support surface chemistry influences on the activation state of RhoA mediated by adsorbed proteins and distinct changes in adherent cell morphology resulting from modulation of this activation state.