The effects of chilling temperatures, in light or dark, on the isolated thylakoids and leaf discs of cucumber (Cucumis sativa L. "Marketer") and spinach (Spinacia okracea L. "Bloomsdale") were studied. The pretreatment of isolated thylakoids and leaf discs at 4 C in the dark did not affect the phenazine methosulfate-dependent phosphorylation, proton uptake, osmotic response to sucrose, Ca2+-dependent ATPase activity, or chlorophyll content. Exposure of cucumber cotyledon discs and isolated thylakoids of cucumber and spinach to 4 C in light resulted in a rapid inactivation of the thylakoids. The sequence of activities or components lost during inactivation (starting with the most sensitive) are: phenazine methosulfate-dependent cyclic phosphorylation, proton uptake, osmotic response to sucrose, Ca2+-dependent ATPase activity, and chlorophyll. The rate of loss of proton uptake, osmotic response to sucrose, Ca2+-dependent ATPase activity and chlorophyH is similar for isolated cucumber and spinach thylakoids, whereas spinach thylakoids are more resistant to the loss of phenazine methosulfate-dependent phosphorylation. The thylakoids of spinach leaf discs were unaffected by exposure to 4 C in light. The results question whether the extreme resistance of spinach thylakoids treated in vivo is solely a function of the chloroplast thylakoid membranes and establish the validity of using in vitro results to make inferences about cucumber thylakoids treated in vivo at 4 C in light.It is well documented that thermophilic plants are damaged by temperatures between 0 and approximately 12 C (chilling injury). Much of the earlier work concentrated on alterations in the fruit (5, 12) and vegetative parts (11, 17) of plants. Less attention has been paid to the leaves of thermophilic plants. Reported alterations in leaf metabolism include effects on the photosynthetic process (4,9,14,15,21, 29), respiration (1,8), activation energy of enzymes (21), and altered membrane permeability (19).The injury due to chilling temperatures is particularly acute in the presence of light. The photosynthetic rates of thermophilic plants (Paspalum, Sorghum, Cucumis, and Glycine) decrease rapidly when the temperature is lowered from 25 to 10 C in the light (9, 25). Most work on the combined effects of light and chilling temperatures has concentrated on CO2 fixation, primarily in C4 grasses (3,24,26), although available data indicate that photochemical activities are altered when leaves are chilled in light (9).