Based on the integral role that argininosuccinate synthase (AS) plays in the production of nitric oxide in vascular endothelial cells and urea in liver, an analysis was carried out to determine whether signals reside in the AS mRNA to account for tissue differences in AS function and location. Reverse transcriptase-PCR and sequence analysis showed that the AS mRNA coding region was the same for both endothelial cells and liver; however, 5'-RACE analysis (rapid amplification of cDNA ends) identified AS mRNA species in endothelial cells in addition to a major 43-nucleotide (nt) 5'-untranslated region (UTR) AS mRNA with overlapping extended 5'-UTRs of 66 and 92 nt. Comparison to the genomic sequence immediately upstream of the reported transcription start site for the human and mouse AS gene suggested that expression of all three species of bovine endothelial AS mRNA are driven by a common promoter and that 5'-UTR diversity in endothelial cells results from three transcriptional initiation sites within exon 1. RNase protection analysis and real-time reverse transcriptase-PCR verified and quantitated the differential expression of the extended 5'-UTR species relative to the major 43-nt 5'-UTR AS mRNA. In vitro translation studies showed a less pronounced but similar discordant expression. Sequential deletions starting from the 5' terminus of the 92-nt 5'-UTR construct resulted in a corresponding increase in translational efficiency, but the most pronounced effect resulted from mutation of an upstream open reading frame, which restored translational efficiency of the 92-nt 5'-UTR AS mRNA. When the different AS mRNA 5'-UTRs, cloned in front of a luciferase reporter gene, were transfected into endothelial cells, the pattern of luciferase expression was nearly identical to that observed for the different 5'-UTR AS mRNAs in endothelial cells. Given the different roles ascribed for argininosuccinate synthase, urea versus NO production, these results suggest that sequence in the AS gene represented by position -92 to -43 nt from the translation start site in the extended AS mRNA 5'-UTRs plays an important role in differential and tissue-specific expression.