We have developed a simple, versatile, and nondisruptive method for isolating basement membranes (BM) from several organ subfractions. In the past, investigators have employed sonication as an almost universal step in the isolation and purification of BM. This procedure, however, frequently results in cellular contamination and fragmentation of the starting material. Our method relies on the selective solubilization of cell membranes, intracellular protein, and plasma proteins with the detergents Triton X-100 and deoxycholate. This technique yields a preparation of BM which is ultrastructurally intact, nonfragmented, and which retains its histoarchitectural relationships. Basement membrane isolated from kidney glomeruli and tubules and brain and retinal microvessels show continuous sheets, free of cellular debris. Interstitial collagens are virtually excluded. The BM from different sources exhibit a wide variation in thickness and morphology emphasizing a nonunitary concept of BM. In all cases, however, isolated BM are ultrastructurally indistinguishable from their in vivo counterparts. Amino acid and carbohydrate analyses of previously well-characterized BM (lens capsule and glomerular BM) show that following isolation by detergents their chemical compositions compare favorably with reported values. Other sources of BM show similar compositions with high contents of glycine, hydroxyproline, and glutamic acid. All BM show nearly equimolar amounts of glucose and galactose while mannose, fucose, and hexosamines are present in smaller quantities. We conclude that our method of BM isolation may be of particular value where studies of both the fine structure and chemical composition of BM are to be examined. © 1978 Academic Press, Inc.