Controlled proteolysis of MuLV gp70s results in the generation of several fragments which correspond to distinct structural domains of the molecules. The orientation of these regions in gp70 was determined by analysis of the immunoreactivities of proteolytic products generated from the MuLV PrENV polyprotein toward monoclonal alpha p15(E) and alpha gp70 antibodies, and by fragmentation analysis of gp70s specifically labeled with [35S]cysteine and [35S]methionine. These studies confirmed our previous assignment of a p15(E)-disulfide-linked 33K fragment to the carboxy terminus of Akv gp70 (Pinter, Honnen, Tung, O'Donnell, and Hammerling, Virology 116, 345-351, 1982). Using similar fragmentation procedures, the sizes and structural features of gp70 domains of Akv and MCF 247 MuLV gp70s were compared. Trypsinization of MCF-247 gp70 resulted in the production of a carboxy terminal fragment which resembled that of the ecotropic gp70 in that (1) it was disulfide linked to p15(E) but not to the amino terminal fragments, (2) reacted with monoclonal antibody 35/56, (3) contained cysteines but no methionines, and (4) carried only endo H-resistant oligosaccharide chains. Amino terminal MCF gp70 fragments were obtained with apparent molecular weights of 42K and 30K, considerably smaller than the corresponding Akv fragments of 49K and 35K. These MCF fragments were much more stable to degradation by trypsin than the Akv amino terminal components, indicating the loss or inaccessibility of several trypsin sites in the MCF amino terminal domain. These results demonstrated the Akv and MCF 247 gp70s contained highly conserved carboxy terminal domains but unique amino terminal sequences. Common features for both gp70s were the presence of an endo H-sensitive oligosaccharide chain near the amino terminus, and the presence of internal disulfide bonds in the amino terminal domains which resulted in an increased mobility for these fragments when analyzed under nonreducing conditions. Thus, while the amino terminal domains of the two gp70s are structurally different, certain aspects of glycosylation specificity and secondary conformation are conserved, suggesting that these structural features may be important for common biological properties of these molecules.