Hepatitis delta virus (HDV) RNA ribozyme system which consists of three RNA oligomer strands (substrate 8-mer; enzyme 16-mer plus 35-mer, Fig. 1) was designed. Effects of Mg2+ concentration on the pseudo first-order rate constant (kobs) of RNA cleavage reaction and on conformation of ribozyme complex were examined. The secondary structure of the complex was also analyzed by limited digestion with ribonucleases. The kobs and CD data were analyzed by curve-fitting analysis using equations derived for two-Mg2+ and three-Mg2+ ion binding models. The result revealed that a three-Mg2+ binding model can explain the Mg(2+)-concentration-dependent changes of both conformation and activity of the HDV ribozyme.