In order to analyze the organization of the membrane proteins pre M, M, and E of the West Nile (WN) flavivirus we have studied the influence of proteolytic cleavage of intact virus on the structure of these proteins. The amino acid sequence of all proteins is known, all six disulfides present in the viral E protein have been identified, and it has been suggested that the E protein contains regions R1, L1, R2, L2, and R3, which together form the E protein ectodomain followed by a carboxyterminal membrane anchor region (Th. Nowak and G. Wengler (1987) Virology 156, 127-137). The results of our analyses can be summarized as follows: (1) The surface of the WN virus contains E protein oligomers; the E protein molecules present in these structures contain two segments which are exposed to proteolytic attack; the segments are located in parts L1 and R3 of the E protein. (2) Proteolytic cleavage of these oligomers in these regions neither destroys nor releases the oligomers from the viral surface. (3) The WN virus surface contains a layer of 7-nm ring-shaped subunits identifiable by electron microscopy which are neither destroyed nor released by proteolytic cleavage. (4) An E protein trimer can be isolated from the surface of protease-treated WN virus. This trimer is morphologically similar to the 7-nm ring-shaped element which can be identified on the surface of native and protease-treated WN virus by electron microscopy.