Cell penetrating peptides (CPPs) are small peptides that are able to penetrate the plasma membrane of mammalian cells. Because these peptides can also carry large hydrophilic cargos such as proteins, they could potentially be used to transport biologically active drugs across cell membranes to modulate in vivo biology. One characteristic feature of the CPPs is that they typically have a net positive charge. Therefore, a key issue associated with the transport mechanism is the role of the transmembrane electrochemical potential in driving the peptides across the membrane. In this study, we have reconstituted bacteriorhodopsin (bR) in large unilamellar vesicles (LUVs) with fluorescein-labeled CPP penetratin enclosed within the LUVs under conditions when the fluorescence is quenched. Illumination of the bacteriorhodopsin-containing LUVs resulted in creation of a transmembrane proton electrochemical gradient (positive on the inside). Upon generation of this gradient, an increase in fluorescence was observed, which shows that the proton gradient drives the translocation of penetratin. The mechanism most likely can be generalized to other CPPs.