A valinomycin-mediated K+ diffusion potential across the membrane of multilamellar liposomes is stable for longer than 30 min and can be collapsed by a nonselective channel such as gramicidin. The kinetics of the potential collapse are complex but can be qualitatively broken down into a series of processes involving (1) binding of the gramicidin to the outer membrane, (2) dimerization to form a functional channel, (3) the flow of ions through the channel, (4) the establishment of a new diffusion potential on the next bilayer within the multilamellar liposome, and (5) the dissociation of gramicidin from the outer bilayer into the adjacent internal aqueous space. These processes are then repeated, in turn, for all the internal bilayers until the K+ concentration gradient (and membrane potential) is completely dissipated. Process 5 appears to be rate limiting at high gramicidin concentrations, but ion flux, process 3, becomes slower at low gramicidin concentrations where the collapse of the K+ gradient displays voltage dependence. Of course the rates of these processes can also be manipulated by changing the composition or size of the liposome and by varying the ion concentrations. Since the diffusion potential can be conveniently monitored with a voltage-sensitive fluorescent dye, 3,3'-diethylthiodicarbocyanine iodide [diS-C2-(5)], a simple method for the detection and partial characterization of membrane pores emerges from this investigation.