A circular form of Bacillus subtilis ribonuclease P RNA (C-P RNA) was generated in vitro by splicing permuted intron-exon (PIE) sequences containing the P RNA sequence. Steady-state cleavage of pre-tRNA(Asp) catalyzed by circular P RNA is slightly faster than the linear form. Furthermore, steady-state turnover catalyzed by circular RNase P RNA is activated by the addition of the Bacillus subtilis protein component of RNase P, to a rate constant equal to the linear holoenzyme under identical conditions. Also, the circles are resistant to nuclease degradation, have less sequence heterogeneity, and may enhance the formation of a unique structure. Therefore, circular forms of RNase P RNA should prove useful for mutagenesis and structural studies.