Murine leukemia virus (MuLV) encodes two independent pathways for expression of the gag gene. One pathway results in processing and cleavage of the precursor Pr65gag to yield the internal capsid proteins of the virion and is analogous to gag polyprotein precursors for all classes of retroviruses. The other pathway, which is not encoded by several other classes of retroviruses, begins with a glycosylated polyprotein gPr80gag . gPr80gag is synthesized independently of Pr65gag; it contains Pr65gag peptides and additional amino-terminal protein. It is modified by further addition of carbohydrate, exported to the cell surface, and released from the cell but does not appear in virus particles. To investigate the role of glycosylated gag in MuLV infection, two mutants of Moloney MuLV (M-MuLV) deficient for synthesis of gPr80gag but able to synthesize Pr65gag were constructed. The mutants were obtained by substitution into a molecular clone of M-MuLV DNA by DNA from two acutely transforming viruses, Ableson MuLV (Ab-MuLV) and Moloney murine sarcoma virus (M-MSV). Both Ab-MuLV and M-MSV are derived from M-MuLV and they express M-MuLV gag sequences, but some strains do not synthesize glycosylated gag protein. For Ab-MuLV, a 177-base-pair Pst I fragment from the P90 strain containing the initiation codon for Pr65gag was substituted for the equivalent fragment in M-MuLV DNA. For M-MSV, 1.5 kilobases at the 5' end of the genome was substituted. Transfection of the recombined DNAs onto NIH-3T3 cells produced infectious M-MuLV, although the infected cells did not produce gPr80gag. Therefore glycosylated gag is not absolutely required for MuLV replication. Deletion of the glycosylated gag pathway did not significantly reduce the level of virus production, although a minor difference in XC plaque morphology was observed.