There are two basically different mechanisms for the fluorescence and absorption changes of merocyanine, cyanine and oxonol dyes. The permeant dyes (cyanine and oxonol dyes, with delocalized charges) work by a potential-dependent accumulation mechanism. These dyes show large (up to 80%) fluorescence and absorption changes with suspensions of cells, and the changes are complete in seconds. The impermeant dyes (merocyanine dyes, with localized charges) and the permeant dyes also show optical changes that take place in fractions of milliseconds. The rapid optical changes are relatively small (less than or equal to 5 X 10(-3)) but can often be easily detected in experiments with single cells. The rapid, nonaccumulative, optical changes result from membrane-localized dye movements. Cyanine dye-absorption changes occur because of a potential-dependent partition of dye between the membrane and the adjacent aqueous region at the high dye-concentration side of the membrane. Dimers and larger aggregates are formed in the aqueous region during the change. Merocyanine dyes may also work by the same mechanism. DiS-C3-(5) is presently the best dye for measuring membrane potentials of cells, organelles, and vesicles in suspension, but several other cyanines work nearly as well (P.J. Sims, A.S. Waggoner, C.-H. Wang, J.F. Hoffman, Biochemistry 13:3315, 1974). For each system, the ratio of dye to membrane must be varied until the optimum fluorescence change is found. A separate calibration curve must be obtained for each system. For measuring fluorescence and/or absorption changes in single cells, merocyanine 540 and diBA-C4-(5) work well but produce some photodynamic damage with high intensity illumination. A rhodanine merocyanine (WW-375) gives very large absorption changes and does not damage tissue during strong illumination. As the mechanisms of the optical changes are worked out, it should be possible to design and synthesize more sensitive, less toxic dyes that are easier to calibrate. And, as the mechanisms of the optical changes are worked out, these dyes may be useful for studying the structure and dynamics of excitable membranes.