Mouse, hamster, rabbit, horse, and human interferons bind to immobilized Cibacron Blue F3GA under appropriate solvent conditions. Three forms of the immobilized ligand have been investigated: Cibacron Blue F3GA-Sepharose 4B, Blue Dextran-Sepharose 4B and Blue Sepharose CL-6B. The strength of binding of an interferon depends critically on the sorbent: Cibacron Blue F3GA-Sepharose 4B is the weakest in the series and Blue Sepharose CL-6B the strongest. The use of Blue Dextran-Sepharose 4B--a sorbent of intermediate binding properties--allows the complete separation of hamster, mouse and human fibroblast interferons in a single chromatographic step. Indeed, both the resolution, as well as the recovery, of those interferons is complete--regardless of the relative complexity of the chromatographed preparation (containing either crude or purified interferons). Thus, these ligands should prove of considerable use when a facile chromatographic evaluation, both qualitative and quantitative of mammalian interferons is required.