All chloroplast 23S ribosomal RNA genes of the unicellular alga Chlamydomonas reinhardtii contain an 888 bp group I intron with an internal open reading frame (ORF). A precursor RNA encompassing the intron with its 5' and 3' flanking sequences was shown to self-splice both during in vitro transcription and upon incubation of the isolated pre-RNA under self-splicing conditions. Expression of the internal ORF in Escherichia coli in the presence of a plasmid containing a cDNA corresponding to the intronless form of the 23S rRNA gene resulted in specific cleavage of the cDNA at or close to the exon junction sequence. To test whether this ORF-encoded double-strand DNA endonuclease is involved in intron mobility in vivo, the same ribosomal cDNA was stably integrated into the C. reinhardtii chloroplast genome using particle gun mediated transformation. All the transformants with the cDNA integrated at the expected site in the chloroplast genome had the intron precisely inserted at the artificial exon junction site. These experiments demonstrate that the chloroplast ribosomal intron of C. reinhardtii behaves as a ribozyme in vitro and also as a mobile genetic element in vivo provided a target site is present.