Across the thylakoid membrane of spinach chloroplasts a diffusion potential was created by rapid changes in the KCl concentration (salt-jumps) employing a flow and mixing technique. The analysis of salt-induced electrochromic absorbance changes indicates that under appropriate experimental conditions the salt-jump technique facititates the establishment of a well-defined thylakoid voltage. This approach appears to be well suited for quantitative studies of electric field effects on thylakoid reactions. In the open reaction center state, for positive salt-induced thylakoid voltages (positive at the lumenal side) the fluorescence yield of spinach thylakoids increases; for negative voltages it decreases. A linear relation between fluorescence yield and thylakoid voltage is observed in the range from −70 mV to +165 mV, characterized by a change of the fluorescence yield by 9% (± 2%) per 100 mV. Analysis of the fluorescence emission spectra reveals that the electric field effect (i) does not originate from a Stark shift of the emission spectrum and (ii) reflects an influence on the Photosystem II (PS II) fluorescence. With increasing degree of PS II trap-closure, the extent of the electric field effect decreases, and in the closed reaction center state no effect is detectable. The trap-closure sensitivity suggests that the electric field effect on the fluorescence yield originates from an electric field dependence of the PS II charge separation/recombination reactions. We propose that the observed fluorescence yield-thylakoid voltage relation reflects the free energy dependence of the rate constants of the PS II charge transfer reactions.