We investigated the catalytic efficiency and the specificity of the Bacillus subtilis RNase P holoenzyme reaction with substrates that contain a single strand, a hairpin loop, or a tRNA 3′ to the cleavage site. At a saturating ribozyme concentration, RNase P can cleave a single-stranded RNA at ∼0.6 min−1 at pH 7.8. Replacing the single-stranded RNA 3′ to the cleavage site by a hairpin loop or by the yeast tRNAPhe increases the cleavage rate by up to ∼600-fold and ∼3,200-fold, respectively. These results show that compared to a single-stranded RNA substrate, the cleavage rate for the holoenzyme reaction is primarily enhanced by an acceptor-stem-like helix. Substrate binding, ∼7–10 μM for a single-stranded RNA, improves by ∼1,000-fold upon the addition of the tRNA. The efficiency of the RNase P holoenzyme cleaving a single-stranded RNA is sufficiently high to consider autolytic processing of the RNase P RNA (denoted P RNA) transcript in the cell. The addition of the RNase P protein to a precursor form of the P RNA in vitro results in autolytic processing of the 5′ and the 3′ end of this precursor in a matter of minutes. Autolytic processing produces the reported 5′ end of the mature P RNA. The precise 3′ end generated by autolytic processing is different over the course of the reaction and the final product is 4 nt shorter than the reported 3′ end of the B. subtilis P RNA. The observed 3′ end in vitro is consistent with the property of the holoenzyme reaction with single-stranded RNA substrates. The discrepancy with the reported 3′ end may be due to other processing events in vivo or inaccurate determination of the mature 3′ end of the P RNA isolated from the cell. We propose that the mature B. subtilis P RNA is generated at least in part by autolytic processing upon the binding of the RNase P protein to the precursor P RNA.