By a mild and highly reproducible fractionation of Torpedo californica electric tissue, we prepared membrane which was 30 times enriched in nicotinic acetylcholine receptor (AChR). This preparation was neither alkali-stripped nor reconstituted and consequently contained nu (43-kDa protein), which is associated with the cytoplasmic aspect of the receptor. We tested this membrane for the presence of sealed vesicles and determined the orientation of these vesicles by combining three methods. Two of these methods were based on the accessibilities, in the presence and absence of detergent, of the extracellular acetylcholine binding site to alpha-bungarotoxin and of the intracellular nu to trypsin. These two methods are specific for AChR-containing membrane. The third method was morphometry of electron micrographs, by which we estimated the proportion of sequestered membrane. These methods taken together indicated that approximately 45% of the AChR-containing membrane was in the form of leaky vesicles or sheets, 33% was sealed right-side-out vesicles, 11% was sealed inside-out vesicles, and 11% was sequestered within multilamellar or multivesicular vesicles. The complexity of this membrane needs to be taken into account in sidedness studies of the AChR.