A radioimmunoassay for human interferon gamma (IFN-gamma) has been carried out using a recombinant glycosylated interferon (Hu IFN-gamma) as tracer, the N.I.H. reference preparation (Gg 23-901-530) and a polyclonal rabbit antiserum. The assay is highly specific for IFN-gamma: there is no cross-reaction either with interferons alpha and beta, Interleukins 1 and 2, tumor necrosis factor alpha and beta or with various brain peptides. The sequential saturation procedure allowed a sensitivity of 0.4 U/ml with intra and between assay coefficients of variation less than 8 and 12%, respectively. The in-vitro production of IFN-gamma by peripheral blood mononuclear cells (P.B.M.C.) was also measured. In unstimulated cultures, IFN-gamma production remained undetectable, i.e. below the 0.4 U/ml sensitivity level. After stimulation of P.B.M.C. from normal subjects with increasing amounts of PHA, both the 3H-thymidine incorporation and IFN-gamma release followed bell-shaped curves. There was no significant difference of 3H-thymidine incorporation between PHA stimulated cultures (0.2 and 2.5 ug/ml) from normal subjects (36 cases) and those with active (16 cases) or non-active (14 cases) rheumatoid arthritis. At two PHA concentrations of 0.2 and 2.5 ug/ml, mononuclear cells from patients with active disease produced significantly less IFN-gamma than those from either controls or cases with non-active disease.