Activity gel analysis of cell extracts from slow- and fast-growing mycobacteria confirmed the presence of several RNase H activities in both classes of organism. The rnhA gene from Mycobacterium smegmatis (Ms) was subsequently cloned using an internal gene segment probe [Mizrahi et al., Gene 136 (1993) 287-290]. The gene encodes a polypeptide of 159 amino acids that shares 50% identity with the RNase HI from Escherichia coli (Ec). However, unlike its counterparts from Gram- bacteria, Ms rnhA does not form an overlapping divergent transcriptional unit with dnaQ (encoding the epsilon (proofreading) subunit of DNA polymerase III). Ms RNase HI was overproduced in Ec as an enzymatically active maltose-binding protein (MBP) fusion protein which cleaved the RNA strand of an RNA.DNA hybrid with a similar site selectivity to that of its Ec homologue.