The region extending from -40 to -54 of the 5'-flanking region of the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene shows homology to sequences found in the 5'-flanking regions of other cytokine genes, those encoding interleukin-4 (IL-4), IL-5, and granulocyte colony-stimulating factor (G-CSF). This sequence element is referred to as conserved lymphokine element 0 (CLE0). Saturation mutagenesis of the CLE0 element indicates that in addition to the previously mapped region between -73 and -91 (CLE2+ GC box), the CLE0 element is necessary for induction of the mouse GM-CSF gene by phorbol myristate acetate/Ca ionophore (A23187) stimulation in T cells. The presence of the CLE0 element is necessary to observe stimulation of the transcription activity of the mouse GM-CSF promoter in vitro. Mobility shift assays revealed that this region forms an inducible DNA-protein complex, NF-CLE0, which consists of two complexes of similar mobility, NF-CLE0a and NF-CLE0b. NF-CLE0a and NF-CLE0b recognize the 3' half and 5' half of the CLE0 element, respectively, with an overlapping region recognized by both proteins. The recognition sequence of NF-CLE0a corresponds to the region required for induction by phorbol myristate acetate/A23187, while the recognition sequence of NF-CLE0b contains bases that have inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)