Trypanoplasma borreli belongs to the bodonid/cryptobiid group of kinetoplastid protozoa, which represents a sister group to the trypanosomatids. RNA transcripts from several mitochondrial genes in this organism undergo the trypanosomatid type of uridine addition/deletion RNA editing. A guide RNA (gRNA) cDNA library was constructed and five gRNAs were identified, one for editing the ribosomal protein S12 mRNA, three for editing the cytochrome oxidase subunit I mRNA, and one for editing the cytochrome b mRNA. All of the gRNAs contained nonencoded oligo[U] sequences at the 3' end, as is common with gRNAs in trypanosomatids, but also contained nonencoded oligo[U] sequences at the 5' end. The mechanism for addition of the 5' nonencoded oligo[U] sequence and the function of this sequence are unknown. The T. borreli gRNAs were shorter (25-35 nt, excluding the 5' oligo[U]) than gRNAs in trypanosomatids (45-50 nt), indicating a smaller size of editing blocks in this organism. Genomic sequences for two gRNAs were cloned and sequenced. These two gRNA-encoding sequences were shown to originate from the 180-kb Component I molecules, which represent a possible homologue of minicircle DNA in trypanosomatids, and not from the 80-kb Component II molecules, which contain the structural genes and cryptogenes.