The putative relationship between the light-induced absorbance increase at 530 nm (ΔA530), the so-called light-scattering change, and non-photochemical chlorophyll fluorescence quenching (NPQ) was examined by the effect of inhibitors. Antimycin at a low concentration (350 nM) completely inhibited fluorescence quenching while only partially inhibiting A530. This effect was independent of the mode of thylakoid energisation and preinduction of violaxanthin de-epoxidation. Dibucaine at 20 FM abolished NPQ but had little effect on ΔA530. Moreover, the light-induced ΔA530 signal was present even in the absence of de-epoxidised xanthophylls. The cation exchanger A23187 blocked the development of NPQ as well as relaxed fluorescence quenching at steady state without involving a major portion of ΔA530. Thus, the relationship between energy-dependent A530 changes and fluorescence quenching was non-linear under all conditions tested. The light-induced absorbance increase at 530 nm, therefore, is insufficient for NPQ. The differential effects of inhibitors are explained schematically, depicting three phases for NPQ: (a) formation of zeaxanthin and antheraxanthin by the xanthophyll cycle; (b) formation of a state reflected by A530 that is induced by the transthylakoid ApH, possibly involving aggregation of LHCII; and (c) fluorescence quenching by the combined effect of both steps and by the H+-cation exchange properties of thylakoid membranes.