The RNA moieties of ribonuclease P purified from both E. coli (Ml RNA) and B. subtilis (P-RNA) can cleave tRNA precursor molecules in buffers containing either 60 mM Mg*+ or 10 mM Mg2+ plus 1 mM spermidine. The RNA acts as a true catalyst under these conditions whereas the protein moieties of the enzymes alone show no catalytic activity. However, in buffers containing 5-10 mM Mg*’(in the absence of spermidine) both kinds of subunits are required for enzymatic activity, as shown previously. In the presence of low concentrations of Mg2+, in vitro, the RNA and protein subunits from one species can complement subunits from the other species in reconstitution experiments. When the precursor to E. coli 4.5 s RNA is used as a substrate, only the enzyme complexes formed with Ml RNA from E. coli and the protein moieties from either bacterial spe ties are active.