Eosinophilia is a uniquely specific phenomenon regulated by interleukin- 5 (IL-5), suggesting specific control for IL-5 gene expression. Using a transient-transfection reporter assay and DNA mobility-shift experiments in EL4 mouse lymphoma cells, reporter expression and binding of transcription factors to the conserved lymphokine element 0 (CLE0) in the mouse (mIL-5) promoter was investigated. Activation of the IL-5 promoter required costimulation of T cells with phorbol ester (phorbol 12-myristate 13-acetate [PMA]) and cyclic adenosine 3′,5′- monophosphate (cAMP), but was blocked by the immunosuppressive drug, cyclosporin A (CsA). Binding to CLE0 was induced under conditions optimal for IL-5 transcription but was not blocked by CsA. CD28-induced signals could partly substitute for cAMP. However, the effects of cAMP, but not of CD28, were sensitive to the cAMP-dependent protein kinase inhibitor, H89, suggesting that CD28 does not involve a cAMP mechanism. It therefore appears that IL-5 expression can be induced by at least two distinct stimulatory pathways. Although CLE0 contains sequences similar to AP-1 and NF-AT, only the AP-1 moiety of the CLE0 element could be demonstrated to have inducible binding. Experiments with antisera to the AP-1 family of transcription factors indicated that c- fos and JunB bind to the IL-5 CLE0 in activated lymphoma cells. The role of the NF-AT-like element was less clear. A constitutively expressed protein showed a weak band that was inhibited by mIL-2 NF-AT competitor sequences. However, this protein did not react with an anti- NF-ATp antiserum. On the other hand, transcription was partially inhibited by an oligonucleotide containing the intact NF-AT-like element from CLE0, suggesting that the element is important for optimal transcription, but the nature of the protein binding to it remains unknown. The fact that these factors are induced in a subclone of EL4 that does not express IL-5 and bind to a number of other cytokine gene promoters suggests that although binding to CLE0 appears to be necessary for IL-5 transcription, other factors must control the specific expression of the gene.