Thylakoid lumen pH measurements were made using the fluorescent dye 8-hydroxypyrene 1,3,6-trisulfonic acid (HPTS; pyranine). The purpose was twofold: (1) to develop the method for use in the fluorescence ratio mode (permitting assays independent of lumenal dye concentration and lumen volume); (2) to use the technique to measure transmembrane ΔpH values in thylakoids predicted by previously used criteria to be energizing ATP formation with a delocalized or localized protonmotive force. The dye was readily loaded into thylakoids at pH 6 and the loading, washing and resuspension steps did not disrupt the localized or delocalized energy coupling patterns, tested by the effect of a permeable amine on the flash number required to initiate ATP formation as described in earlier work (Beard, W.A. and Dilley, R.A. (1988) J. Bioenerg. Biomembr. 20, 129). Light-dependent dye fluorescence changes were recorded in continuous illumination over 10–20 s with low- or high-salt-stored thylakoids under either coupled or basal conditions to assess the extent of the ΔpH developed across the membranes. Low-salt stored membranes - which were predicted by the criteria developed earlier to show localized Δ \\ ̃ gm H + energy coupling - gave a ΔpH below the thermodynamically predicted energetic requirement for ATP formation at external pH of 7.8 to 8.9. At an external pH of 7.8 or 8.0 the lumen pH under coupled conditions in the low-salt-stored thylakoids was about 6.8 to 7.0, respectively, ( ΔpH ≈ 1.0 unit), and at pH 8.2 the ΔpH was about 1.4 units (lumen mpH = 6.8). At pH 8.9 the lumen pH reached near 7.3 (ΔpH 1.6) under the coupled conditions for low-salt-stored thylakoids. Under basal conditions (no ADP) low-salt-stored membranes gave a ΔpH of 2.4 to 2.8 units, at moderate and high intensities, respectively, when the external pH was 8.9. The pH calibration is not sensitive enough at pH values less than about 6.0 to 6.3 to obtain accurate internal pH data in that range. For that reason, when the external pH was 7.8 to 8.0 all that can be said is that the basal ΔpH could be observed to be >1.7 units. At an external pH of 8.9, high-salt-stored thylakoids - predicated to have a delocalized Δ \\ ̃ gm H + coupling mode - showed a μpH >2.3 in all cases, coupled or basal, but in the coupled mode the ΔpH was near 2.3 at low light and near 2.7 at high light intensity. That is the expected result of the faster electron-proton transport at the high intensity. These lumen pH measurements support the concept developed with other techniques that thylakoids can maintain either localized or delocalized energy coupling Δ \\ ̃ gm H + gradients, and that in the localized energy coupling mode, the H + ions involved in driving ATP formation do not equilibrate with the lumen.