To assess the potential value of ribozyme mediated inhibition of caspase 3 in protection against apoptotic cell death, three M1RNA GSs against human caspase 3 mRNA were designed and synthesized by PCR using the catalytic RNA subunit of RNase P as a template, and their cleavage activities in vitro and in vivo were determined. The 32 P labled caspase 3 transcript being target RNA, cleavage reaction in vitro showed that pM1 GS716 and pM1 GS337 were active, the extent of pM1 GS716 cleavage was 93%. The ribozyme pM1 GS716 constructed could downregulate the expression of caspase 3 in vivo and protect HeLa cells from apoptosis induced by TNF α. Caspase 3 mRNA expression was reduced by 75% and caspase 3 protein was reduced by 69% in HeLa cells transfected with pM1 GS716 by quantitative RT PCR and Western blot analyses, respectively. Caspase 3 protease activity was reduced by 52% by ELISA. Moreover, similar decreased level of apoptotic cells was assessed by staining with Hoechst 33258 compared with control exposure to TNF α(21.6±0 7% vs 49 4±0 2%, P <0 01). These results confirm that pM1 GS716 prepared in vitro possesses the perfect specific catalytic cleavage activity, and is hopeful to effectively inhibit apoptosis in vivo through cleaving the key gene, caspase 3.