The observed levels of Delta G(ATP) in chloroplasts, as well as the activation behavior of the CF(1)CF(0)-ATP synthase, suggest a minimum transthylakoid proton motive force (pmf) equivalent to a Delta pH of approximately 2.5 units. If, as is commonly believed, all transthylakoid pmf is stored as Delta pH, this would indicate a lumen pH of less than approximately 5. In contrast, we have presented evidence that the pH of the thylakoid lumen does not drop below pH approximately 5.8 [Kramer, D. M., Sacksteder, C. A., and Cruz, J. A. (1999) Photosynth. Res. 60, 151-163], leading us to propose that Delta psi can contribute to steady-state pmf. In this work, it is demonstrated, through assays on isolated thylakoids and computer simulations, that thylakoids can store a substantial fraction of pmf as Delta psi, provided that the activities of ions permeable to the thylakoid membrane in the chloroplast stromal compartment are relatively low and the buffering capacity (beta) for protons of the lumen is relatively high. Measurements of the light-induced electrochromic shift (ECS) confirm the ionic strength behavior of steady-state Delta psi in isolated, partially uncoupled thylakoids. Measurements of the ECS in intact plants illuminated for 65 s were consistent with low concentrations of permeable ions and approximately 50% storage of pmf as Delta psi. We propose that the plant cell, possibly at the level of the inner chloroplast envelope, can control the parsing of pmf into Delta psi and Delta pH by regulating the ionic strength and balance of the chloroplast. In addition, this work demonstrates that, under certain conditions, the kinetics of the light-induced ECS can be used to estimate the fractions of pmf stored as Delta psi and Delta pH both in vitro and in vivo.