Concanavalin A (Con A) affinity chromatography was used to concentrate and partially purify a murine gamma interferon (MuIFN gamma) from large volumes of crude MuIFN gamma (specific activity (SA), 10(4) units/mg of protein) produced by mitogen-induced T lymphocytes. Elution of the MuIFN gamma from the immobilized Con A with alpha-methyl-D-mannoside resulted in a 13-fold purification. Further purification of the Con A-bound MuIFN gamma was accomplished by gel filtration chromatography (SA 10(6.7) units/mg of protein) which also demonstrated the molecular weight (MW) to be 38,000 +/- 3000. Molecular weight determinations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and nonreducing conditions, revealed that the Con A-bound MuIFN gamma may exist as a dimer, consisting of monomers of 20,500 MW. Specific rabbit antiserum raised against the Con A-bound MuIFN gamma neutralized the antiviral activities of crude, Con A-bound and unbound MuIFN gamma fractions, as well as the 20,500 MW MuIFN gamma.