We previously isolated cDNA and genomic DNA of the mouse glial cell line-derived neurotrophic factor (GDNF) gene and found that the gene consists of three exons. Recently, it was suggested that an alternative promoter exists within intron 1 of the human GDNF gene, but this has not been confirmed. Novel cDNA clones of the mouse GDNF gene were isolated by 5'-rapid amplification of cDNA ends from postnatal day-14 striatum. A novel exon, containing 351 nucleotides, exists between exon 1 and exon 3 (referred to as exon 2 in our previous report). Luciferase reporter assay showed that a core promoter for the novel exon 2 requires its 5'-untranslated region. Primer extension analysis and reverse transcription-PCR identified another novel transcript that starts 39 bp upstream of exon 3, and the core promoter activity exists within a region containing putative Sp1 sites. Although the core promoters for the novel exons are different from those previously identified, transcripts derived from each promoter coincidentally increased with interleukin-1beta or tumor necrosis factor-alpha stimulation. Gel retardation assays suggested that the NF-kappaB binding site in intron 1 would be involved in the cytokine response of the mouse GDNF gene.