Hepatitis 8 virus (HDV) contains a singlestranded circular RNA genome of 1.7 kilobases. In this report we demonstrate that subfragments of HDV RNA can undergo autocatalytic cleavage. This cleavage requires at least 500 pM of Mg2+ or Ca2+, is not affected by varying the pH from 5.0 to 9.1, and occurs with RNA fragments as small as 133 nucleotides. The larger RNA fragments containing additional HDV sequences have a lower efficiency of cleavage. Deletion analysis at both ends of RNA subfragments suggested that the catalytic ability of HDV RNA resides in a stretch of no more than 117 nucleotides around the cleavage site. The cleavage occurs at the phosphodiester bond between nucleotides 688 and 689 on the HDV genomic map, generating a 5' fragment with a terminal uridyl 2',3'-cyclic monophosphate residue and a 3' fragment with a guanosyl residue with a 5'-hydroxyl group. The smallest autocleaving RNA does not contain the "hammerhead" sequence required for the autocleavage of other known selfcleaving RNA. The cleavage of HDV RNA occurs at a much faster rate, even at a very low Mg2+ concentration, than that of other "ribozymes." Thus, HDV RNA represents a distinct class of ribozyme.Human hepatitis 8 virus (HDV) is a defective virus often associated with fulminant hepatitis in hepatitis B virus carriers. The virus particles have an envelope in which hepatitis B virus surface antigen is located. Internal to the envelope are HDV-specific 6 antigen and a single-stranded circular RNA genome of 1.7 kilobases (kb) (1-3). This animal virus is distinctive because it has such a mixed structure that resembles somewhat the virus coat protein-encapsidated plant virus satellite and virusoid RNAs. The HDV RNA possesses several additional properties similar to those of circular RNA of viroids; for instance, the presence of a high degree of intramolecular self-complementarity and conservation of the consensus sequences known to be important for viroid RNA replication (4,5). However, HDV RNA is much larger than viroid RNAs and encodes at least one structural protein (6 antigen), whereas viroid RNA does not encode any protein. Despite these differences HDV RNA and viroid RNA appear to replicate through a similar mechanism-i.e., a rolling circle mechanism (6)-since a larger-than-genomic size RNA intermediate has been detected in HDV-infected cells (7,8). Thus a mechanism must exist to cleave these RNA intermediates into monomeric RNA and to circularize them.One viroid and several virusoid and satellite RNAs have been shown to possess an autocleaving activity (9-11). The cleavage sites of these RNAs correspond to sites of in vivo RNA processing, suggesting that these autocleavage reactions have physiological roles. A linear dimer cDNA of HDV genome can be processed into a monomeric RNA when transfected into a monkey kidney (COS) cell line (unpublished observation), suggesting either that HDV RNA has an unusual secondary structure that allows for specific attack by a cellular RNase or that HDV RNA has a specific autocleavage activit...