Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.