Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3'-5' exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E coli, but not in higher organisms, and close homologues are present in other genomes from the beta and gamma subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (k(cat) = 100-650 min(-1), K-M = 0.4-2.0 mM, at pH 8.00 and 25 degrees C, with 1 mM Mn2+). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn2+ being preferred over Mg2+ as cofactor, and was inhibited by Ni2+. The concentration dependency of Mn2+ was examined, giving K-Mn values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.