The structural organization of the lymphocytic choriomeningitis virus (LCMV) particle has been examined by Triton X-114 phase separation and nearest neighbor analyses in order to define protein-protein interactions in the virion. Extraction with Triton X-114 established that the 44-kDa membrane glycoprotein, GP-1, is a peripheral protein and that the 35-kDa glycoprotein, GP-2, is an integral membrane protein. Membrane permeable and membrane impermeable crosslinking reagents were used to establish the structural organization of the virion. Results obtained with both types of crosslinking reagents demonstrated that both GP-1 and GP-2 were assembled as native homotetramers. No covalent or disulfide linkages were found between GP-1 and GP-2, nor were these glycoproteins crosslinked. Protein complexes composed of GP-2 and NP were observed after treatment with a membrane permeable crosslinker (DMS) but not after treatment with the membrane impermeable crosslinker (DTSSP), localizing the site of the GP-2:nucleocapsid protein (NP) interaction to the interior of the virion. The interaction of GP-2 with NP may be important in directing the maturation and budding of LCM virions.