Immunoprecipitation of labeled extracts from murine leukemia virus-infected cells with antisera specific for internal structural (gag) proteins yields three major gag-related polyproteins with molecular weights of 180,000 (Pr180gag-pol), 80,000, and 65,000 (Pr65gag). It has been shown by others that Pr65gag is the immediate precursor of the internal structural (gag) protein, and that Pr180gag-pol is the precursor to reverse transcriptase. In studies reported here, the 80,000-dalton gag-related polyprotein from Moloney strain murine leukemia virus (M-MuLV)-infected cells was found to be glycosylated by the following criteria: (i) incorporation of [3H]mannose, (ii) a change in electrophoretic mobility upon digestion with endoglycosidase H, and (iii) a change in electrophoretic mobility when glycosylation was inhibited by treatment of the cells with tunicamycin during labeling. The 80,000-dalton gag polyprotein has therefore been designated GpP80gag. The unglycosylated form of GpP80gag was a polypeptide of 75,000 daltons. A comparison of [3H]mannose and [3H]galactose labeling experiments suggested that GpP80gag is further glycosylated to yield a glycopolypeptide of 95,000 daltons. This 95,000-dalton polypeptide is relatively rapidly cleaved to yield two glycopeptides of 55,000 and 40,000 daltons which are released into the cell culture fluid, as soluble proteins. Cell-free translation of M-MuLV genomic RNA resulted in two major gag-related products of 75,000 and 65,000 daltons. The 65,000-dalton gag-related cell-free translation product comigrated with Pr65gag, and the 75,000-dalton cell-free product comigrated with the unglycosylated form of GpP80gag. Both of the gag-related cell-free translation products could be labeled with [35S]formyl methionine, which is incorporated only as the N-terminal amino acid during translation. Other investigators have shown that GpP80gag and Pr65gag differ at their N-termini, and these results combined with those reported here suggest that GpP80gag and Pr65gag are translated from two separate initiation sites in M-MuLV RNA.