Inhibition of heat shock protein 90 (Hsp90) has emerged as a novel intervention for the treatment of solid tumors and leukemias. Here, we report that F1F0-ATP synthase, the enzyme responsible for the mitochondrial production of ATP, is a co-chaperone of Hsp90. F1F0-ATP synthase co-immunoprecipitates with Hsp90 and Hsp90-client proteins in cell lysates of MCF-7, T47D, MDA-MB-453, and HT-29 cancer cells. Inhibition of F1F0-ATP synthase by efrapeptins results in the disruption of the Hsp90 complexing with its substrate proteins and, in most cases, in the degradation of the latter. Hsp90-client proteins affected by the inhibition of F1F0-ATP synthase included ER alpha, mutated p53 (m.p53), Hsp70, Hsp27, and caspase-3 but not Raf-1. This is the first report identifying caspase-3 as a substrate protein,of Hsp90. Unlike typical Hsp90 inhibitors, efrapeptin treatment triggers Hsp70 downregulation in parallel with depletion of Hsp90. This suggests that suppression of Hsp90 chaperone function through inhibition of F1F0-ATP synthase does not result in activation of transcription factor HSF-1, a generally unfavorable consequence of anti-cancer treatments based on Hsp90 inhibition. (c) 2006 Elsevier Inc. All rights reserved.