The effects of cations on the structure and activity of isolated barley (Hordeum vulgare cv. Svalöfs Bonus) chloroplast lamellar systems were studied. Three separate effects of cations were recognizable. (1) Cations in high concentrations (in excess of 100–200 mM NaCl) are required to maintain granal stacks once the outer chloroplast envelope is broken. In 30 mM NaCl the majority of the lamellar systems exist as well-separated thylakoids, even in the presence of high concentrations of sucrose (330 and 660 mM). The lamellar systems photoreduce ferricyanide at coupled rates whether they are in the granal or non-granal configuration. (2) Cations are required for Hill reaction activity. This requirement becomes apparent following loss of thylakoid membrane integrity at very low cation concentrations. Maximum activation of the photoreduction of ferricyanide occurs at 30 mM NaCl, and divalent ions (Mg++, Ca++, Mn++) are 12 times more effective than monovalent ions (Na+, K+). (3) Cations are necessary to preserve the integrity of thylakoid membranes. Below about 8 mM NaCl, the thylakoids swell and this change is accompanied by progressive loss of Hill activity and the capacity to maintain a light-dependent proton gradient. Divalent cations are more than 200 times as effective as monovalent ions in preventing these changes. Hill reaction activity, but not proton pump activity, is regained by adding cations back to the swollen thylakoids (cation effect 2, above) and the new rate of Hill reaction activity is the same as in chloroplasts uncoupled with methylamine. The re-establishment of Hill reaction activity is accompanied by appression of the swollen thylakoids. The uncoupler methylamine causes stacking of thylakoids and collapsing of intrathylakoidal spaces.