The binding of lipophilic cation probes of membrane potential to cells was re-examined. Even concentrations of probe molecules as low as 100 nM were found to reduce delta psi and thus many commonly used techniques for delta psi determination are inappropriate. Binding was found to be a linear function of probe concentration and independent of pH. The proportionality constant for binding has been equated to an "apparent binding volume' for [3H]TPP+ with units of microliter/mg dry weight of cells. This "apparent binding volume' is thermodynamically equivalent to the volume of cell membrane multiplied by the partition coefficient of [3H]TPP+ for cell membrane and was equivalent to 9.10 +/- 0.33 microliters/mg dry weight in Enterococcus faecalis. It was concluded that the most accurate method for delta psi determination was to use nanomolar concentrations of lipophilic cations and appropriate correction for energy dependent binding.