The unique hormonal characteristics of human interleukin 2 (IL 2), primarily the high affinity of the IL 2-receptor interaction, created several impediments to the generation of monoclonal antibodies to this lymphokine. Because normal cell sources produce only a few micrograms of IL 2 per liter, it was necessary to utilize high producer clones and subclones of a human T leukemia cell line to obtain immunogenic amounts of IL 2 protein. Moreover, assays that required antibody-mediated intervention of the high affinity IL 2-receptor binding were ineffectual in the identification of anti-IL 2-producing hybridomas, thus necessitating the development of immunoassays. Two of three initially derived antibodies detected by enzyme-linked immunoassay were found to react specifically with IL 2 as demonstrated by antibody concentration-dependent neutralization of IL 2 activity. The neutralization of cellular proliferation was specific for IL 2-reactive cells, coincided with an inhibition of IL 2 receptor binding, could be completely overcome by affinity-purified IL 2 and was species-specific; human and murine IL 2 were neutralized, whereas rat IL 2 activity remained unaffected. A third antibody, although much less effective in neutralizing IL 2 activity, bound to IL 2 more avidly and functioned as an efficient immunoabsorbent. IL 2 could be concentrated and purified by immunoabsorption from crude conditioned medium in a single step. The purified product, which retained biologic activity, was made up of a single protein (Mr = 15,500) as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed-phase liquid chromatography, and amino-terminal amino acid sequence analysis. These results indicate that the distinctive biochemical and functional properties of individual lymphokines may well determine the efficiency with which antibodies may be elicited and detected. However, once produced, anti-lymphokines are uniquely suited for the exploration of the molecular and biologic properties of these immunoregulatory molecules.