Ribozymes have been developed to cleave the bcr-abl transcripts and thereby suppress transforming activities of chronic myelogenous leukemia (CML) cells. However, the intracellular efficacy of vector-dependent ribozymes usually depends in part on their expression cassettes, which may affect their intracellular trafficking and distribution. In order to test effects of an intron in pre-fusion ribozyme on the anti-bcr-abl activities in CML cells, retroviral vectors harboring ribozyme expression cassettes with (RzI) or without (Rz) an intron-encoding sequence were used to transduce K562 cells. In terms of both reduction of the target bcr-abl mRNA and suppression of colony formation in soft agar and xenograft growth on SCID mice, the anti-bcr-abl efficacy of the RzI fusion ribozyme was significantly superior to that of Rz. These results also correlate with more cytoplasmic accumulation of the RzI fusion ribozymes than that of the Rz. This study suggests activities of a RNA polymerase II-driven fusion ribozyme against its targeted spliced mRNA are improved by incorporating an intron in its pre-splicing transcript. Noticeably, the improvement is contributed in part by subcellular co-localization.