RNA interference appears to be a potentially powerful tool for studies of genes of unknown function. However, differences in efficacy at different target sites remain problematic when small interfering RNA (siRNA) is used as an effector. Similar problems are associated with attempts at gene inactivation using antisense oligonucleotides (ODNs) and ribozymes. We performed a comparative analysis of the suppressive effects of three knockdown methods, namely, methods based on RNA interference (RNAi), antisense ODNs, and ribozymes, using a luciferase reporter system. Dose-response experiments revealed that the IC50 value for the siRNA was about 100-fold lower than that of the antisense ODN. Our results provide useful information about the positional effects in RNAi, which might help to improve the design of effective siRNAs.