We report the cloning of the RNase P RNA genes from the primary aetiological agent of porcine pneumonia, Mycoplasma hyopneumoniae, and the closely related commensal, Mycoplasma flocculare. The monocistronic genes each have promoters with AT-rich -35 regions and Rho-independent-like transcription terminators which are retained in the RNase P RNA. Both of these RNase P RNA variants are shown to be catalytically active in vitro in spite of a low overall GC content (30%). Our results suggest a new example of a stable mini-helix in the conserved core of the mycoplasmal RNase P RNAs. Deletion of the corresponding structural element in Escherichia coli RNase P RNA (M1 RNA) generated an RNase P RNA with an impaired substrate interaction. Displacement of this structural element with the mycoplasmal mini-helix resulted in an enzyme with a phenotype similar to that of wild-type M1 RNA. In addition, this structural element is important for lead ion-induced cleavage at specific sites in M1 RNA.