We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine-treated glass (PLglass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially adsorbed. First, monolayer freeze-fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface), in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic side by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.KEY WORDS purple membrane fracture polylysine cationic ferritin membrane asymmetry freeze-The plasma membrane of the halophilic bacterium Halobacterium halobiurn contains differentiated patches (1) that can be isolated by hypotonic cell lysis followed by density gradient centrifugation (24,27). The membrane fragments are purple with a broad absorption maximum around 570 nm, and contain a single 26,000-dalton protein, bacteriorhodopsin (1,3,22), 75% by weight, and several classes of glyco-, sulfo-, and phospholipids (15, 16), 25% by weight. Two sulfated lipids (glycolipid sulfate and phosphatidyl glycerosulfate) are found exclusively in the purple membrane (PM) fraction whereas the dominant phospholipid (phosphatidyl glycerophosphate) is also present in the red plasma membrane fraction (15,16). Upon absorption of light, bacteriorhodopsin undergoes a photoreaction cycle (17, 18) that involves the uptake and release of hydrogen ions (14,17,18,23). There is good evidence that ion release is vectorial (14,17,18), generating a proton gradient that can be used as an energy source (25) consistent with the chemiosmotic mechanism for energy transduction (20,23). Although the bulk lipid composition of the PM has been well studied (15, 16) and the threedimensional structure of the bacteriorhodopsin J. CELL BIOLOGY 9 The Rockefeller University Press 9