Murine interferon-gamma (MuIFN-gamma), produced by the T-cell lymphoma, L12-R4, and stimulated with phorbol myristic acetate, was purified by rat monoclonal antibodies AN-18.17.24. The purified product retained its biologic activity and consisted of two proteins (Mr = 17,500 and 16,500), as determined by sodium dodecyl sulfate-gel electrophoresis. Both species were found to be glycosylated, since their Mr values decreased to 14,500 and 13,500 when the producing cells were stimulated in the presence of tunicamycin. Analysis of intracellular and secreted forms in the presence of [35S] methionine revealed that MuIFN-gamma is synthetized as a single peptide of Mr = 17,500 and undergoes proteolytic cleavage during or after secretion. Peptide mapping by reverse phase high pressure liquid chromatography showed indeed that the peptide profile of both species is very similar, suggesting a high degree of homology in the primary structure. These results demonstrate that the molecular heterogeneity of the MuIFN-gamma is probably the outcome of proteolytic processing during or after secretion.