Non-photochemical fluorescence quenching (NPQ) in diatoms is associated with a xanthophyll cycle involving diadinoxanthin (DD) and its de-epoxidized form, diatoxanthin (DT). In higher plants, an obligatory role of de-epoxidized xanthophylls in NPQ remains controversial and the presence of a transthylakoid proton gradient (ΔpH) alone may induce NPQ. We used inhibitors to alter the amplitude of ΔpH and/or DD de-epoxidation, and coupled NPQ. No ΔpH-dependent quenching was detected in the absence of DT. In diatoms, both ΔpH and DT are required for NPQ. The binding of DT to protonated antenna sites could be obligatory for energy dissipation.