Recently, we have reported that the nuclear large subunit pre-rRNA of the green alga Chaetomorpha moniligera has an intron. Now we report that the intron in this pre-rRNA self-splices in vitro and that its structure constructed shows that it is one of the group I B introns. The self-splicing reaction of this intron is inhibited by gentamycin or L-arginine. The intron consists of 653 nucleotides. The primary structure of the intron was derived from the sequence of a region of 25S rDNA that contains the intron. Its 5` terminal base is cytosine and 3` terminal base of the 5` exon that is linked to the intron is uracil. On the other hand, on the basis of tripartite structure that is proposed to form for the selection of 3` splice site, 3` terminal base of the intron has been inferred to be guanine on the position 653. The 5` terminal base of the 3` exon that is linked to this guanine is uracil. Like the other group I introns, the intron in pre-rRNA of Chaetomorpha moniligera contains nine base-paired stems P1 through P9, and it contains also four conserved sequence elements in the following order: 5`-P(GAAUGCGGGAGC)-Q(CCUCCGCAGG)-R(CCCCUAGAGA-CUAGA)-S(AUUCGAGAGUCCU)-3`. Major distinct features of this intron differing from most of the other group I introns are that it has an extra stem P6c which is formed between 3` half of the stem P6a and 3` half of the stem P6, and that the stem P8 has a long extension which contains an open reading frame.