The interleukin-2 (IL-2) assay has become a useful tool for examining the cellular immune response. Because of a lack of avian IL-2-dependent cell lines, the avian IL-2 assay in its present state, however, is not currently as powerful as its mammalian counterpart in effectively evaluating levels of IL-2 activity. The use of an avian IL-2 reference standard, Percoll-enriched populations of lymphoblasts, and sample collection times were examined to optimize the assay for comparing levels of IL-2 activity in a large number of birds. The reference standard was effective in accounting for assay-to-assay variance. Percoll-enriched populations of responding lymphoblasts were more sensitive to IL-2 than nonseparated populations of cultured peripheral blood lymphocytes. Levels of IL-2 activity of cells isolated from the same bird did not change within a 1-week period, although differences did approach significance. In addition, levels of IL-2 activity differed between genetically distinct populations.