A DNA cassette system has been developed to allow for the convenient introduction of synthetic DNA oligonucleotides between the transcription and translation start sites of a gene in order to examine the effect of 5' hairpin structure and strength on mRNA stabilization. Rationally designed synthetic DNA cassettes were introduced into the 5' untranslated region of a modified lacZ gene to form hairpins at the 5' end of the mRNA. These DNA inserts influenced mRNA half-lives over an order-of-magnitude range, with some groups of predicted structures having half-lives that showed a strong correlation with hairpin strength while half-lives for another group of predicted structures exhibited little or no dependence on this property. These results indicate the importance of 5' hairpin structure and strength in determining stabilization of Escherichia coli mRNA. This synthetic library, as well others generated using the DNA cassette system described here, should prove useful in understanding the mechanisms of mRNA stabilization and in designing structures for recombinant gene expression control.