The expression of glutamine synthetase (GS), catalysing the ATP-dependent conversion of glutamate and ammonia into glutamine, is transcriptionally and post-transcriptionally regulated. The genomic structure of dog GS shown in the present study is basically similar to that of other mammals in that it is composed of seven exons and six introns. Using 5'-cRACE (where cRACE stands for circular rapid amplification of cDNA ends) and reverse transcriptase-PCR, we identified an additional exon (120 bp) in the first intron, designated in the present study as exon 1'. By means of alternative splicing. the GS gene produces an altered form of GS transcript with 5'-untranslated region (UTR) containing the exon 1'. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. In the human and mouse GS genes, extra exons are also found at the corresponding site of the intron 1 but with different sizes. An exon-trapping experiment for the GS gene in COS-7, Madin-Darby canine kidney and SK-N-SH cells revealed that the pattern of alternative splicing is variable in different cell types. The propensity of forming a secondary structure is predicted to be considerably higher in the presence of extra 5'-UTR, suggesting the possibility of a translational effect. To test this, we performed a reporter assay for fusions with different 5'-UTRs, demonstrating that the long form with extra 5'-UTR was translated 20-and 10-fold less than the short one in SK-N-SH and Neuro-2A cells respectively. Similarly, translations of human and mouse transcripts with extra 5'-UTRs were less efficient, showing 6-8-fold reductions in SK-N-SH cells. Furthermore, when we mutated an ATG sequence contained in the exon 1', the suppression of translation was partially relieved, suggesting that the negative regulation by an extra 5'-UTR is, to some extent, due to an abortive translation from the upstream ATG.