The great sensitivity of the luciferin-luciferase ATP detection system allows direct observation of ATP formation derived from single-turnover flashes in a thylakoid reaction mixture. The method can measure the energization threshold--the number of flashes required for the initiation of ATP formation--as well as detect post-illumination ATP formation after the last flash of a flash sequence. We describe the characteristics of this post-illumination phosphorylation which can be observed after a series of phosphorylating flashes (PIP+) or when the assay for ATP formation was performed in a "traditional" manner where the ADP and Pi were added after the flash-energization period (PIP-). Comparing PIP+ yields and kinetics of the PIP+ decay under various treatments can give information about membrane energization events only if it is clearly established that different PIP+ yields and decay rates are not due to limitations of the luciferase-catalyzed reaction. Experiments showing that the PIP+ ATP yield and kinetics were due to membrane-limited deenergization events (proton efflux) rather than luciferase limitations include: (1) An uncoupler, nigericin, added after the last flash reduced the PIP+ yield, but had no effect on the luciferase reaction. (2) The kinetics of the luminescence after adding standard ATP were much faster than the PIP+ kinetics. (3) Valinomycin and K+ stimulated the PIP+ yield but had no influence on the luciferase reaction. (4) Lowering the pH from 8 to 7 increased both the PIP- (an assay independent of luciferase kinetics) and the PIP+ ATP yields, an expected result owing to the greater endogenous buffering power encountered by the proton gradient when the external pH is 7. In spite of the last-mentioned point, the threshold flash number for ATP formation onset was the same for pH 7 and 8 (valinomycin, K+ present) at slow flash frequencies. This is consistent with a membrane-localized rather than a delocalized gradient. The accompanying reports (W. A. Beard, G. Chiang and R. A. Dilley, and W. A. Beard and R. A. Dilley, J. Bioenerg. Biomembr.) show that different conditions can lead to observing either localized or delocalized proton gradient coupling in the PIP+ event and the ATP onset threshold flash number.