Proteinase K treatment of the bovine brain acetylcholinesterase (AChE) releases a hydrophobic fragment of 13 kDa, which is entirely responsible for the aggregation of the G4 AChE in the absence of detergent. This observation provides evidence that the 13 kDa fragment, which comes from a previously identified 20 kDa subunit, is directly involved in the attachment of the G4 AChE to brain membranes. A model for the organization of the different sub-domains of the hydrophobic anchor of the G4 AChE is presented.