Cyanobacteria contain a bidirectional [NiFe] hydrogenase which transiently produces hydrogen upon exposure of anoxic cells to light, potentially acting as a “ valve ” releasing excess electrons from the electron transport chain. However, its interaction with the photosynthetic electron transport chain remains unclear. By GFP-tagging the HoxF diaphorase subunit we show that the hydrogenase is thylakoid associated, comprising a population dispersed uniformly through the thylakoids and a subpopulation localized to discrete puncta in the distal thylakoid. Thylakoid localisation of both the HoxH and HoxY hydrogenase subunits is con fi rmed by immunogold electron microscopy. The diaphorase HoxE subunit is essential for recruitment to the dispersed thylakoid population, potentially anchoring the hydrogenase to the membrane, but aggregation to puncta occurs through a distinct HoxE-independent mechanism. Membrane association does not require NDH-1. Localization is dynamic on a scale of minutes, with anoxia and high light inducing a signi fi cant redistribution between these populations in favour of puncta. Since HoxE is essential for access to its electron donor, electron supply to the hydrogenase depends on a physiologically controlled localization, potentially o ff ering a new avenue to enhance photosynthetic hydrogen production by exploiting localization/aggregation signals. hydrogenase interacts with the electron transport chain. We investigated hydrogenase location and behaviour in the model unicellular cyanobacterium Synechocystis sp. PCC 6803, location having a direct bearing on access to electron donors. We demonstrate that the hydrogenase has two distinct physiologically-controlled localization mechanisms that partition it within the thylakoids, either dispersed uniformly through the thylakoids or aggregated into discrete puncta. Crucially, electron supply and hydrogen production depend on localization. Determination of the molecular basis for control of hydrogenase location could thus pave the way to engineering improved cyanobacterial cells for solar-powered bio-hydrogen production.