The TCATTT-containing element extending from -61 to -41 of the mouse IL-5 gene is highly conserved in the corresponding region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and has been previously shown to be involved in regulating inducible GM-CSF gene expression. By using stable transfection assays in the mouse Th2 clone D10.G4.1, we show that the TCATTT-containing element is also involved in the regulation of inducible IL-5 gene expression. The mouse IL-5 and GM-CSF homologues of this element were found by gel shift analysis to form DNA-nuclear protein complexes of similar electrophoretic mobility under conditions in which expression of these genes is induced. However, comparative studies using extracts of D10.G4.1 cells treated with the cellular activators Con A and PMA and the inhibitors cycloheximide and cyclosporin A indicated that the binding activities to the conserved elements in the IL-5 and GM-CSF genes (designated NF-IL-5A and NF-GM-CSFA, respectively) are regulated by different signaling pathways. In addition, NF-IL-5A is not induced in the Th1 clone HDK-1 which does not express the IL-5 gene. The strong correlation between the signal-dependent and cell-specific modulation of IL-5 and GM-CSF gene expression patterns and the binding activities of NF-IL-5A and NF-GM-CSFA suggests that these nuclear proteins are involved in the transduction of T cell activation signals to the transcriptional machinery of these genes through their interactions with their respective TCATTT-containing elements.