tRNase Z, which exists in almost all cells, is believed to be working primarily for tRNA 3' maturation. In Escherichia coli, however, the tRNase Z gene appears to be dispensable under normal growth conditions, and its physiological role is not clear. Here, to investigate a possibility that E. coli tRNase Z cleaves RNAs other than pre-tRNAs, we tested several unstructured RNAs for cleavage. Surprisingly, all these substrates were cleaved very efficiently at multiple sites by a recombinant E. coli enzyme in vitro. tRNase Zs from Bacillus subtilis and Thermotoga maritima also cleaved various unstructured RNAs. The E. coli and B. subtilis enzymes seem to have a tendency to cleave after cytidine or before uridine, while cleavage by the T. maritima enzyme inevitably occurred after CCA in addition to the other cleavages. Assays to determine optimal conditions indicated that metal ion requirements differ between B. subtilis and T. maritima tRNase Zs. There was no significant difference in the observed rate constant between unstructured RNA and pre-tRNA substrates, while the K(d) value of a tRNase Z/unstructured RNA complex was much higher than that of an enzyme/pre-tRNA complex. Furthermore, eukaryotic tRNase Zs from yeast, pig, and human cleaved unstructured RNA at multiple sites, but an archaeal tRNase Z from Pyrobaculum aerophilum did not.