The plastid (cyanelle) from the Glaucocystophyceae alga Cyanophora paradoxa contains an RNase P RNA subunit (P RNA) similar to the cyanobacterial P RNA. We have synthesized this RNA by in vitro transcription and analyzed its activity in the absence or presence of the RNase P protein subunit (P protein) from Escherichia coli and the cyanobacterium Synechocystis sp. PCC 6803. In contrast to the bacterial P RNA, the cyanelle P RNA is not active in the absence of protein in any of the conditions tested. A functional enzyme could be reconstituted with the Synechocystis protein but not with the E. coli protein. This is the first demonstration of RNase P activity reconstitution from organellar and bacterial subunits.