Previously, we reported that pooled normal human serum contained anti-IL-1 alpha autoantibodies. Further characterization studies have been undertaken with sera from individual healthy humans. Molecular exclusion chromatography demonstrated that 9 of 38 test sera contained anti-IL-1 alpha autoantibodies that specifically bound 125I-IL-1 alpha. The autoantibodies formed immune complexes composed of either one IgG or two IgG molecules bound to one 125I-IL-1 alpha molecule. The data suggest that the autoantibodies recognize at least two separate antigenic sites on IL-1 alpha. The autoantibodies neutralized IL-1 alpha induced IL-2 secretion by mouse thymocytes (EL-4 NOB-1). Further, in an in vitro competitive receptor binding assay, the autoantibodies completely blocked 125I-IL-1 alpha binding to recombinant human IL-1 receptor expressed on CHO cells. Our previous studies have established a correlation between inhibition of 125I-IL-1 alpha binding to receptor bearing cells and neutralization of IL-1 bioactivity in vitro and in vivo. These data suggest that development of anti-IL-1 alpha autoantibodies may play a significant role in modulating the effects of high local or systemic concentrations of IL-1 alpha.