We have constructed a strain (CT1) that expresses RNase P conditionally with the aim to analyze the in vivo tRNA processing pathway and the biological role that RNase P plays in Synechocystis 6803. In this strain, the rnpB gene, coding for the RNA subunit of RNase P, has been placed under the control of the petJ gene promoter (P(petJ)), which is repressed by copper, cell growth, and accumulation of RNase P RNA is inhibited in CT1 after the addition of copper, indicating that the regulation by copper is maintained in the chimerical P(petJ)-rnpB gene and that RNase P is essential for growth in Synechocystis. We have analyzed several RNAs by Northern blot and primer extension in CT1. Upon addition of copper to the culture medium, precursors of the mature tRNAs are detected. Furthermore, our results indicate that there is a preferred order in the action of RNase P when it processes a dimeric tRNA precursor. The precursors detected are 3'-processed, indicating that 3' processing can occur before 5' processing by RNase P. The size of the precursors suggests that the terminal CCA sequence is already present before RNase P processing. We have also analyzed other potential RNase P substrates, such as the precursors of tmRNA and 4.5 S RNA. In both cases, accumulation of larger than mature size RNAs is observed after transferring the cells to a copper-containing medium.