We present a double-antibody radioimmunoassay for determination of the concentration of tumor necrosis factor (TNF) in serum. TNF in serum competes with a fixed amount of 125I-labeled TNF for the binding sites of specific rabbit antibodies. The bound TNF is precipitated with Sepharose-bound anti-rabbit IgG, then centrifuged, and the radioactivity of the pellets is counted. The detection limit of the assay is 7 ng/L (B0-3 SD). Bound radioactivity in the range of 10% to 90% of the B0 counts corresponds to TNF concentrations of 26 to 10,000 ng/L. Of 40 sera from healthy subjects, 21 (53%) contained TNF concentrations greater than 7 ng/L (range 8-40 ng/L). Some patients with parasitic or neoplastic disease and patients with septic shock had highly increased TNF values. Three of the 14 sera (21%) from patients with rheumatoid arthritis had TNF concentrations greater than 40 ng/L.