To study the effect of specific G11 nucleotide on cleavage activity of hairpin ribozyme in vitro, the 32P-labeled pKC transcript (267 nt) containing hepatitis B virus core region is used as target RNA. Synthetic hairpin ribozyme genes with G11 mutational site are cloned into ribozyme vector p1.5 to create pHpRz for preparation of ribozyme. Non-labeled HpRz transcripts are incubated with 32P-labeled target-RNAs under different conditions and autoradiographed after gel electrophoresis. The results show that the cleavage activity of hairpin ribozyme is closely related to the matching of the bases between hairpin ribozyme and the substrate. The G11 site is not essential to cleavage activity of hairpin ribozyme in vitro, so the selection of target sequence matching G11 site of hairpin ribozyme is not limited to this site.