One major determinant of rapid mRNA decay is the presence of AU-rich sequences located in 3' untranslated regions (UTR). To assess for the contribution of upstream sequences on the activity of the 3' AU-rich destabilizing element, we have determined the decay-rates of v-/c-fos hybrid transcripts by quantitative RNA protection analysis. In a transient expression assay, v-/c-fos recombinants generated two mRNA populations via alternative splicing and removal of an optional intron entirely located in the 5' UTR. Both mRNA species were found to be relatively stable in constructs lacking the c-fos AU-rich destabilizing element. Unexpectedly, in recombinants where intrinsic AU sequences were kept intact, only the full-length mRNA population showed high instability whereas the spliced mRNA species remained relatively stable. A v-/c-fos 5' UTR fragment encoding the optional intron was inserted into alpha-globin genes harboring either the c-fos or GM-CSF destabilizing element. The splicing and degradation patterns of these heterologous transcripts paralleled that of v-/c-fos recombinants. These observations unmasked a 5' cis-acting element in v-/c-fos mRNA whose presence is required for the activity of the AU-rich destabilizing element. They demonstrate the important role of interactions between distinct sequences on the regulation of mRNA stability.