We have examined the effects of heat stress on electron transfer in the thylakoid membrane of an engineered plastid ndh deletion mutant, Δ1, incapable of performing the Ndh-mediated reduction of the plastoquinone pool in the chloroplast. Upon heat stress in the dark, the rate of PSII-independent reduction of PSI after subsequent illumination by far-red light is dramatically enhanced in both Δ1 and a wild-type control plant (WT). In contrast, in the dark, only the WT shows an increase in the reduction state of the plastoquinone pool. We conclude that the heat stress-induced reduction of the intersystem electron transport chain can be mediated by Ndh-independent pathways in the light but that in the dark the dominant pathway for reduction of the plastoquinone pool is catalysed by the Ndh complex. Our results therefore demonstrate a functional role for the Ndh complex in the dark.