The nonmevalonate pathway of isoprenoid biosynthesis in the apicoplast of the human malaria parasite Plasmodium falciparum is distinct from the mevalonate-dependent pathway of humans and thus a good drug target. We describe here the hammerhead ribozyme based cleavage of the ispH (lytB) gene transcript involved in the last step of this nonmevalonate pathway. Using RNA folding program, three hammerhead ribozymes named as RZ(876), RZ(1260), and RZ(1331) were predicted against ispH (lytB) mRNA. Messenger walk screening (RNaseH) assay confirmed the target accessibility for these ribozymes. All three ribozymes cleaved the target RNA in vitro but RZ(876) exhibited the highest catalytic potential (62.92%). Therefore, RZ(876) was chemically synthesized with appropriate chemical modifications to protect it from nuclease attack while using it for in vitro parasite growth inhibition assay. This ribozyme RZ(876) was able to inhibit 87.36% parasite growth at 30 microM concentration compared to the untreated culture. However, an absolute inhibition of 29.41% was achieved compared to the control ribozyme (RZ(ctrl)). Nonetheless, the growth inhibition effect was found to be sequence-specific as indicated by the decreased level of ispH (lytB) transcript after ribozyme treatment. In conclusion, we have identified the ispH (lytB) as a potential target whose transcript can be cleaved by a ribozyme RZ(876).