A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA. This RNA encodes a peptide tag that is incorporated at the end of the aberrant polypeptide and targets it for proteolysis. We have identified a circularly permuted version of the tmRNA gene in alpha-proteobacteria as well as in a lineage of cyanobacteria. The genes in these two groups seem to have arisen from two independent permutation events. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. The mature two-piece tmRNAs are predicted to have a tRNA-like domain and an mRNA-like domain similar to those of standard one-piece tmRNAs, with a break located in the loop containing the tag reading frame. A related sequence was found in the mitochondrial genome of Reclinomonas americana, but only the tRNA-like portion is retained. Although several sequence and structural motifs that are conserved among one-piece tmRNAs have been lost, the alpha-proteobacterium Caulobacter crescentus produces a functional two-piece tmRNA.