To identify the divalent metal ions that can support the self-cleavage activity of the genomic ribozyme of human hepatitis delta virus (HDV), we tested the activity of various divalent metal ions in the ribozyme reactions catalyzed by HDV88 (683-770 nt) and 88DI3 (HDV88 with the sequence from 740-752 nt deleted). Among various metal ions tested, Mg2+, Mn2+, Ca2+ and Sr2+ efficiently supported the self-cleavage reactions of the HDV88 and 88DI3 ribozymes. In the case of the 88DI3 ribozyme, other divalent metal ions, such as Cd2+, Ba2+, Co2+, Pb2+ and Zn2+, were also able to support the self-cleavage reaction to some extent (< 10%). In the presence of spermidine (0.5 mM), the cleavage reaction was promoted at lower concentrations of effective divalent metal ions. The HDV ribozyme represents the only example of ribozyme to date of a ribozyme that catalyzes the self-cleavage reaction in the presence of Ca2+ ions as efficiently as it does in the presence of Mg2+ ions.