Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G-protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK₁₀, and measured the activity of RhoA, Rac1 and Cdc42 using GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion so that by 60 minutes there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity, and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 minutes of depletion, but then increased from 30-90 minutes of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia, and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations.