The selective degradation of messenger RNAs enables cells to regulate the levels of particular mRNAs in response to changes in the environment.Ribonuclease (RNase) E , a single-strand-specific endonuclease [2-4] that is found in a multi-enzyme complex known as the 'degradosome' [5-7], initiates the degradation of many mRNAs in Escherichia coli [3,8,9]. Its relative lack of sequence specificity and the presence of many potential cleavage sites in mRNA substrates [2,3] cannot explain why mRNA decay frequently proceeds in a net 5'-to-3' direction [9-11]. I have prepared covalently closed circular derivatives of natural substrates, the rpsT mRNA encoding ribosomal protein S20  and the 9S precursor to 5S ribosomal RNA [1,12], and find that these derivatives are considerably more resistant to cleavage in vitro by RNase E than are linear molecules. Moreover, antisense oligo-deoxynucleotides complementary to the 5' end of linear substrates significantly reduce the latter's susceptibility to attack by RNase E. Finally, natural substrates with terminal 5'-triphosphate groups are poorly cleaved by RNase E in vitro, whereas 5' monophosphorylated substrates are strongly preferred (compare with ). These results show that RNase E has inherent vectorial properties, with its activity depending on the 5' end of its substrates; this can account for the direction of mRNA decay in E. coli, the phenomenon of 'all or none' mRNA decay, and the stabilization provided by 5' stem-loop structures [14-17].