A rapid procedure for isolation of two biologically active human interferon-gamma subtypes was developed. Crude interferon-gamma produced in a serum-free culture of peripheral blood mononuclear cells by mitogen stimulation was concentrated and partially purified by chromatography on controlled-pore glass. Following desalting and concentration by ultrafiltration, a step of cation-exchange high-performance liquid chromatography was performed. A linear NaCl gradient (0.01-0.4 M) at pH 7 was employed and four peaks of biological activity eluting at 0.17, 0.20, 0.26 (major peak), and 0.3 M were obtained. The major peak of biological activity coincided with two protein peaks. Analysis of one fraction from the major activity peak by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a protein band having an apparent molecular weight of 26,000, while an adjacent fraction of the same activity peak contained a protein band corresponding to a molecular weight of 21,000. The specific activity of both subtypes was 7-10 X 10(6) units/mg.