Natural murine interferon-gamma (IFN-gamma) produced by the T lymphoma, L12R4, stimulated with phorbol myristic acetate was purified by anti-murine IFN-gamma immunoadsorbent and labeled with 125I to study its binding to receptors of mouse cells. By adding increasing concentrations of unlabeled natural murine IFN-gamma in competition binding assays we determined a KD of 8.2 X 10(-10) M for L1210 cells and a number of receptors of about 3000. Moreover protease, but not endoglycosidase treatment of target cells prevented the subsequent binding of the ligand. These studies demonstrate that natural murine IFN-gamma binds in a specific manner and with high affinity to receptors on murine cell lines.