We describe a novel DNA and RNA found in the mitochondria of the Varkud-1c natural isolate of Neurospora. The majority of the RNA, termed VSRNA, is an 881 nucleotide single-stranded circular molecule complementary to one strand of a low copy, double-stranded circular DNA, VSDNA. VSRNA combines some features of the human hepatitis delta virus, group I introns, retroelements, and plant viral satellite RNAs. VSRNA synthesized in vitro performs a self-cleavage reaction whose products terminate with a 5' hydroxyl and a 2',3' cyclic phosphate. This reaction may be involved in the natural processing pathway of multimeric VSRNA in vivo. VSRNA lacks a hammer-head structure or substantial sequence similarity to any other self-cleaving RNA, suggesting that the RNA structure involved in cleavage may be different from those in previously characterized catalytic RNAs.