The pH gradient, delta pH, and the membrane potential, delta psi, formed during light-induced electron transport in Rhodospirillum rubrum chromatophores were measured by two independent methods: (a) using specific electrodes to monitor light-dependent uptake of NH4Cl and SCN- at chromatophore concentrations of about 0.1 mg bacteriochlorophyll/ml and (b) using 9-aminoacridine and 8-anilinonaphthalenesulfonic acid as fluorescent probes for delta pH and delta psi, respectively, at chromatophore concentrations of about 0.01 mg bacteriochlorophyll/ml. The light intensity was measured and set at a level which saturated the highest bacteriochlorophyll concentration used. The steady-state values obtained with each method under phosphorylating conditions were compared with the phosphorylation potential maintained by the chromatophores under identical conditions. The results indicate that under all conditions employed the ratio H+/ATP is greater than 2, and varies between 2.4 and 3.4 depending on the method used for estimation of the electrochemical proton gradient.