Membrane-potential-dependent accumulation of diS-C 3 (3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, max , caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red max shift depends on relative cell-to-probe concentration ratio, a maximum shift (572<582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (c5 10 6 cells/ml) and probe (10 7 ) concentrations at which a clearly defined relationship exists between the max shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0•2% glucose (cell wall thickness 0•175 0•015 m, n=30) are stained much faster and the max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0•260 0•043 m, n=44). At a suitable cell and probe concentration and under standard conditions, the max shift of diS-C 3 (3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.