A method for the preparation of plasma membrane fractions from liver is described. A crude membrane fraction, substantially free of mitochondria and nuclei, is collected by centrifuging liver homogenate over a pad of 1.45 M sucrose. This interfacial fraction is further purified by isopycnic centrifugation in a self-generating density gradient of Percoll. All steps are carried out in buffered, isotonic solutions. Membrane prepared in this way is enriched in marker enzymes associated with plasma membrane, while marker enzymes for cellular components other than plasma membrane are diminished. Contamination by mitochondrial components is very much less than for membrane prepared from hypotonic liver homogenates. The membrane fraction also shows specific insulin binding, and negligible degradation of insulin. The procedure is rapid, uses simple equipment, and can be completed within 3 h.