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vacB, a gene previously shown to be required for expression of virulence in Shigellaand enteroinvasive Escherichia coli, has been found to encode the 3′–5′ exoribonuclease, RNase R. Thus, cloning of E. coli vacBled to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy ofvacBeliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacBgene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacBbe renamed rnr.