The chloroplast large subunit rRNA gene of Chlamydomonas eugametos and its 5' flanking region encoding tRNA(Ile) (GAU) and tRNA(Ala) (UGC) have been sequenced. The DNA sequence data along with the results of a detailed RNA analysis disclosed two unusual features of this green algal large subunit rRNA gene: (1) the presence of six group I introns (CeLSU.1-CeLSU.6) whose insertion positions have not been described previously, and (2) the presence of three short internal transcribed spacers that are post-transcriptionally excised to yield four rRNA species of 280, 52, 810 and 1720 nucleotides, positioned in this order (5' to 3') in the primary transcript. Together, these RNA species can assume a secondary structure that is almost identical to that proposed for the 23 S rRNA of Escherichia coli. All three internal transcribed spacers map to variable regions of primary sequence and/or potential secondary structure, whereas all six introns lie within highly conserved regions. The first three introns are inserted within the sequence encoding the 810 nucleotide rRNA species and map within domain II of the large subunit rRNA structure; the remaining introns, found in the sequence encoding the 1720 nucleotide rRNA species, lie within either domain IV or V, as is the case for all other large subunit rDNA introns that have been documented to date. CeLSU.5 and CeLSU.6 each contain a long open reading frame (ORF) of more than 200 codons. While the CeLSU.6 ORF is not related to any known ORFs, the CeLSU.5 ORF belongs to a family of ORFs that have been identified in Podospora and Neurospora mitochondrial group I introns. The finding that a polymorphic marker showing unidirectional gene conversion during crosses between C. eugametos and Chlamydomonas moewusii is located within the CeLSU.5 ORF makes it likely that this intron is a mobile element and that its ORF encodes a site-specific endonuclease promoting the transfer of the intron DNA sequence.