The resistance of certain tumor cells to the chemotherapeutic agent L-asparaginase has often been found to be associated with the presence of asparagine synthetase activity. In an attempt to study the translational regulation of the asparagine synthetase gene, the 5'-untranslated region of human asparagine synthetase cDNA was mapped by antisense oligonucleotide-mediated hybrid arrest translation in reticulocyte lysate. Three consecutive cis-acting regulatory elements, spanning from -60 to -120 bases from the initiation codon, in the 5'-untranslated region of the asparagine synthetase gene, were identified. T1 RNase footprinting analysis showed that those regulatory elements can be protected from T1 digestion when incubated with reticulocyte lysate. A 46-kDa trans-acting protein factor that interacts with the cis-acting regulatory element of asparagine synthetase mRNA was detected. This 46-kDa protein factor is most likely to be the eucaryotic peptide chain initiation factor eIF-4A as determined by immunoprecipitation experiments using a monoclonal antibody raised against reticulocyte eIF-4A.