The chloroplasts contain an extensive system of internal membranes or thylakoids in which all the light-harvesting and energy-transducing processes of the photosynthesis are located. Thylakoids are differentiated into stacked membrane regions (or grana thylakoids) and nonstacked membranes (or stroma thylakoids), each with a specialized structure and function. Both kinds of thylakoids can be separated by detergent-based methods or mechanical fragmentation such as sonication. We describe the fractionation of thylakoid membranes into grana and stroma thylakoids by treatment with the detergent digitonin and successive ultracentrifugation of the resultant vesicles. After their separation, the thylakoid fractions retain electron transport and enzymatic activities and are characterized using various parameters. The stroma thylakoids have higher chlorophyll a/chlorophyll b and protein/total chlorophyll ratios, and greater photosystem I and NADH dehydrogenase activities than the grana thylakoids. In the conditions used and on a protein basis of total thylakoids, the yield of stroma thylakoids is 5%, which is considerable taking into account that the stroma thylakoids are a minor component of total thylakoids.