The magnitude of delta psi (membrane potential), delta pH (pH gradient), lactose accumulation and cytoplasmic volume have been determined over a range of experimental conditions. A study of two probes of delta pH, benzoate and dimethyloxazolidene-2,4-dione (DMO), and four probes of delta psi, Rb+, K+, tetraphenylphosphonium (TPP+) and 3,3'-dipropylthiodicarbocyanine iodide, has been carried out. Benzoate and DMO are shown to be equivalent at pH values above the pK of DMO, but the latter may be less accurate below this pH. The cations TPP+ and Rb+ were found, by a number of criteria, to be equivalent, and TPP+ may be used in cells not pretreated with EDTA. These studies are an essential prerequisite to the use of TPP+ as a quantitative probe in untreated cells.