The fluorescence of the voltage sensitive dye, diS-C3-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C3-(5) fluorescence from cells and from the supernatant. It has been found that diS-C3-(5) fluorescence in the supernatant can be selectively monitored at lambda exc = 630 nm and lambda em = 650 nm, while the cell associated fluorescence can be observed at lambda exc = 690 nm and lambda em = 710 nm. A modified theory for the diS-C3-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, 1n I/I degrees, and the underlying change in the plasma membrane potential, delta psi p = psi p - psi p degrees. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K+ gradients. It has been demonstrated that the membrane potential change, delta psi p, can be measured on an absolute scale.