We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by electron tomography of high-pressure-frozen/freeze-substituted intact chloroplasts. Higher-plant thylakoids are differentiated into two interconnected and functionally distinct domains, the photosystem II/light-harvesting complex II-enriched stacked grana thylakoids and the photosystem I/ATP synthase-enriched, nonstacked stroma thylakoids. The grana thylakoids are organized in the form of cylindrical stacks and are connected to the stroma thylakoids via tubular junctions. Our data confirm that the stroma thylakoids are wound around the grana stacks in the form of multiple, right-handed helices at an angle of 20°to 25°as postulated by a helical thylakoid model. The junctional connections between the grana and stroma thylakoids all have a slitlike architecture, but their size varies tremendously from approximately 15 3 30 nm to approximately 15 3 435 nm, which is approximately 5 times larger than seen in chemically fixed thylakoids. The variable slit length results in less periodicity in grana/stroma thylakoid organization than proposed in the original helical model. The stroma thylakoids also exhibit considerable architectural variability, which is dependent, in part, on the number and the orientation of adjacent grana stacks to which they are connected. Whereas some stroma thylakoids form solid, sheet-like bridges between adjacent grana, others exhibit a branching geometry with small, more tubular sheet domains also connecting adjacent, parallel stroma thylakoids. We postulate that the tremendous variability in size of the junctional slits may reflect a novel, active role of junctional slits in the regulation of photosynthetic function. In particular, by controlling the size of junctional slits, plants could regulate the flow of ions and membrane molecules between grana and stroma thylakoid membrane domains.The term thylakoid is the name coined by Menke (1962) to describe the internal photosynthetic membranes of chloroplasts. Most of our knowledge of the three-dimensional (3D) architecture of higher-plant thylakoids is based on the analysis of electron micrographs of thin-sectioned in situ and isolated chloroplasts (Staehelin, 2003). The first electron micrographs of thin-sectioned chloroplasts in which the internal thylakoid membranes were clearly resolved indicated that they were organized in the form of flat sheets and membrane stacks (Hodge et al., 1955;Steinmann and Sjö strand, 1955). Menke (1960) subsequently postulated that higher-plant chloroplasts contained many individual, sac-like thylakoids of two types, small thylakoids that gave rise to the grana stacks, and large thylakoids (now called stroma thylakoids) that interconnected the grana stacks. Menke's (1960) model was challenged by Weier and coworkers (Weier, 1961;Weier et al., 1963), whose micrographs showed that the stro...