Autoantibodies to various cytokines have been reported in normal individuals and in patients with various infectious and immunoinflammatory disorders, and similar antibodies (Ab) may be induced in patients receiving human recombinant cytokines. The clinical relevance of these Ab is often difficult to evaluate. Not only are in vitro neutralizing cytokine Ab not necessarily neutralizing in vivo, but assays for binding and neutralizing Ab to cytokines are often difficult to interpret. For example, denaturation of immobilized cytokines in immunoblotting techniques and immunometric assays may leave Ab to the native forms of the mediators unrecognized. On the other hand, Ab may bind nonspecifically and/or with biologically irrelevant low affinities, leading to erroneous interpretations. This article describes in detail the use of radioimmunoassays that we have optimized and used successfully for the detection of high-affinity (auto)Ab to IL-1α, IL-6, GM-CSF, and IFNα.