Previous studies have shown that urine of febrile patients contains a tumor necrosis factor a inhibiting activity (TNF-a Inh) when tested in a cytotoxicity assay using the tumor necrosis factor a (TNF-a)-susceptible cell line L929. In the present study, we investigated the relationship between the TNF-a Inh and a potential soluble form of the receptor, as the former has been shown to block TNF-a activities by binding to the ligand. We demonstrate that human TNF-et is affected to a greater extent than is murine TNF-a. This species specificity of the inhibitor correlates with the binding studies of TNF receptor interactions already reported. We raised a polyclonal antibody to TNF-a Inh that neutralizes its activity and does not recognize TNF-a. Solubilized cross-linked '25I-labeled TNF-a receptor complex could be immunoprecipitated by using either anti-TNF-a or anti-TNF-a Inh antibody, suggesting immunological cross-reactivity between the receptor and the inhibitor. By using fluorescein isothiocyanate-coupled TNF-a, it was possible to visualize by fluorescence-activated cell sorter analysis the TNF-a receptor on phytohemagglutinin/interleukin 2-activated T cells. A similar increase of immunofluorescence intensity of the activated T cells was observed by using anti-TNF-a Inh antibody revealed with a fluorescein isothiocyanatecoupled goat anti-rabbit IgG1 conjugate, suggesting that the TNF-a Inh is also expressed as a membrane protein. Taken together, our results suggest that the TNF-at Inh originally described might be a soluble form of the TNF receptor itself.