The putative function of highly conserved regions (HCRs) within 3' untranslated regions (3'UTRs) as regulatory RNA sequences was efficiently and quantitatively assessed by using modular retroviral vectors. This strategy led to the identification of HCRs that alter gene expression in response to oxidative or mitogenic stress. Databases were screened for UTR sequences of >100 nucleotides that had retained 70% identity over more than 300 million years of evolution. The effects of 10 such HCRs on a standard reporter mRNA or protein were studied. To this end, we developed a modular retroviral vector that can allow for a direct comparison of the effects of different HCRs on gene expression independent of their gene-intrinsic 5'UTR, promoter, protein coding region, or poly(A) sequence. Five of the HCRs tested decreased mRNA steady-state levels 2- to 10-fold relative to controls, presumably by altering mRNA stability. One HCR increased translation, and one decreased translation. Elevated mitogen levels caused four HCRs to increase protein levels twofold. One HCR increased protein levels fourfold in response to hypoxia. Although nonconserved UTR sequences may also have a role, these results provide evidence that sequences that are highly conserved during evolution are good candidates for RNA motifs with posttranscriptional regulatory functions in gene expression.