The time-specific requirement of Rho proteins for the S phase progression of vascular endothelial cells was determined by reversibly introducing inhibitor proteins with a cell-penetrating peptide. We found evidence of the reversibility of protein transduction. The removal of extracellular protein caused the transduced protein to decay in a manner sensitive to low temperatures. The time required for a 50% decay correlated with the protein size. The time-specific transduction of the inhibitor proteins uncovered a critical period requiring Rho proteins in the G(1)-S transition phase. Reversible protein transduction may thus be a powerful tool to investigate the time-specific role of signaling proteins. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.