1. Six rat liver plasma-membrane subfractions of different density and morphological, enzymic and chemical properties were prepared from homogenates by a combination of differential, rate-zonal and density-gradient centrifugation. They consisted of three vesicular 'light' subfractions of density 1.12-1.13 and three 'heavy' subfractions of density 1.16-1.18 containing membrane strips and intercellular junctions. 2. All six subfractions contained a basal adenylate cyclase activity. One of the 'light' subfractions that showed the highest glucagon-stimulated adenylate cyclase activity was identified as deriving form the blood-sinusoidal face of the hepatocyte. This subfraction, unlike the others, was contaminated by Golgi components, as indicated by its morphological properties and the presence of galactosyl- and sialyl-transferase activities. 3. All the six subfractions showed high activities of the following plasma-membrane marker enzymes: 5'-nucleotidase, alkaline phosphodiesterase (nucleotide pyrophosphatase), alkaline phosphatase, leucine naphthylamidase and Mg2+-activated adenosine triphosphatase. A 'light' subfraction that showed the highest specific activities of all the above marker enzymes, but lacked a glucagon-stimulated adenylate cyclase activity, was identified as deriving from the bile-canalicular face of the hepatocyte. 4. The 'heavy' subfractions, which showed generally the lowest activities of the above plasma-membrane enzyme markers, and were characterized by the presence of desmosomes and gap junctions, were taken to originate from the contiguous faces of the hepatocyte. 5. The protein composition of the six subfractions was generally similar, as shown by polyacrylamide-gel electrophoresis. Differences in the amounts of various protein and glycoprotein bands among the subfractions correlated with their morphology, enzymic composition and sialic acid content. 6. Hormonal and histochemical evidence supporting the identification of a bile-canalicular subfraction, a blood-sinusoidal subfraction and contiguous-face subfractions is discussed
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