Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein+ (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5-3.0 kV/cm, 40 microseconds) in medium containing 25-50 micrograms/ml PI at 21-23 degrees C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form > 95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic "pulse medium," the membranes resealed after electropulsing with a time constant (tau R) of about 2 min. In 150 mOsm medium, resealing was faster (typically tau R approximately 0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius dependence of pulse-induced voltage (CVradius approximately 10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (Cc), was taken into account. Evaluation of the Cc values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation.