Abstract Three hybridoma cell lines secreting monoclonal IgG antibodies specific for human tumor necrosis factor-binding protein I (TNF-BP I), the extracellular domain of the 60 kDa TNF receptor, were developed by fusion of spleen cells from mice immunized with TNF-BP I purified from urine. The antibodies recognize three different epitopes on TNF-BP I. Two of the antibodies were used to develop a two-site (‘sandwich’) enzyme immunoassay with horseradish peroxidase as the marker enzyme. The assay was able to measure TNF-BP I in serum, urine and cell culture supernatants with a sensitivity of about 200 ng/1 and a precision better than 10%. TNF-BP I was detected in the serum of healthy individuals at a mean concentration of 2.1±1.0 μg/l(mean± standard deviation; range, 0.52–5.4 μg/l, n=42); no significant difference was seen in patients with chronic polyarthritis (2.3±0.79 μg/l; n=15). Serum TNF-BP I was significantly elevated in patients with burns (6.5±1.7 μg/l; n=10) and markedly increased in patients with renal failure (49±17 μg/l; n=6). TNF-BP I was also detectable in urine from normal individuals (2.2±1.2 μg/l; range 0.78−4.3 μl; n=16). Culture supernatants of several human tumor cell lines also contained TNF-BP I. The assay will be a useful tool to detect activation of the TNF receptor by the physiological ligands, TNF-α and TNF-β, as well as transmodulation by other mediators in various pathological conditions.