We previously cloned mouse glial cell line-derived neurotrophic factor (GDNF) cDNA and genomic DNA and found that the mouse gene contains a 1086-bp 5'-untranslated region (5'-UTR). We investigated the contributions of the 5'-UTR to promoter activity and found one positive regulatory region and two negative regulatory regions in the 5'-UTR. In the present study, using gel retardation assays and mutation analyses, two novel cis-elements that interact with nuclear extracts from mouse astrocytes were identified. The first cis-element (nucleotides (nt) +70 to +81) enhances promoter activity, whereas the second cis-element (nt +239 to +247) attenuates promoter activity in a position- and orientation-dependent manner. Suppression of gene expression by a third region (nt +509 to +580) occurs at the translational level. The ATG sequence (nt +547 to +549) has the potential to initiate translation and to attenuate the efficiency of translation for the GDNF precursor coding region. Furthermore, we identified an alternative promoter in the 5'-UTR that is driven by an Sp1 element, circumventing the translational suppression. Taken together, the 5'-UTR of mouse GDNF contains two novel cis-elements, a short upstream open reading frame and an alternative promoter that influences gene expression at both the transcriptional and translational levels.