The transthylakoid pH difference (ΔpH) following shuttered periods of actinic light from 10 ms to 30 s duration was estimated from fluorescence quenching of N-(1-naphthyl)ethylenediamine or 9-aminoacridine in phosphorylating conditions, ± valinomycin and ± nigericin. ΔpH in controls varied from about 2 units after 10 ms to a maximum of 3.6 - 3.8 units in the steady state. The transthylakoid potential difference (ΔV), defined as the average electric potential energy for protons moving from the intrathylakoid spaces to the outside, was estimated from the size of ΔA520-500 (the electrochromic shift) during similar light periods, with a correction for diffuse-double-layer potentials on the outside and inside of the thylakoids. A relation between ΔA520-500 and ΔV followed from the assumption that the former signal during a single-turnover flash represented 30 mV between the insides of the thylakoid surfaces. On this basis ΔV rose rapidly to about 80 mV at ~25 ms, and declined thereafter, to reach a steady-state level of ~13 mV at 10-30 s. The total proton motive force (PMF) thus estimated rose to about 225 mV within 20 ms and remained constant (controls) or declined slowly (+nigericin). With added valinomycin + K+, the PMF rose from 120 mV at 10 ms to c. 190 mV in the steady state, corresponding to low values of ΔV throughout. The findings are discussed in relation to present concepts of electron transport, proton translocation and charge accumulation in chloroplast thylakoids, especially on the millisecond timescale, and compared with other estimates of ΔpH and ΔV.