Plastids, a wide family of plant specific organelles, exist in all plant cells in a number of different forms with different functions essential for plant life. Among them, chloroplasts are by far the more extensively studied owing to their central role in photosynthesis. However, other plastid family members, often referred to as nongreen plastids, play also major roles in the physiology of higher plants and could be better suited for studies of specific metabolic processes in heterotrophic plant cells. Unfortunately, serious technical problems are frequently encountered with separating intact, active nongreen plastids from contaminating membranes and mitochondria. Here, we provide detailed protocols suitable for the large scale preparation of intact and highly pure proplastids from cauliflower buds, as well as amyloplasts from sycamore cultured cells, and for the subsequent separation of their surrounding envelope membranes from the stroma and other plastid fractions. Both methods proved to be highly reliable and have been instrumental for in-depth investigations on biochemistry and physiology of nongreen plastids.