Respiratory syncytial virus (RSV), which presents the primary cause of bronchiolitis and pneumonia among infants and causes significant morbidity and mortality in immunodeficient patients, remains a health problem worldwide. Unfortunately, an effective vaccine is currently unavailable and pharmacologic treatment needs further optimization for RSV disease. Because RSV is a non-segmented negative-strand RNA virus, it may be sensitive to the genome RNA cleaving by DNAzyme, an artificial nucleic acids molecule with high catalytic capability of cleaving complementary RNA molecules. Thus, RSV-targeted DNAzymes potentially present as a therapeutic candidate of RSV diseases. In this study, DNAzymes targeting the RSV genomic RNA or mRNA were designed and synthesized, one of which (DZn1133) did cleave RSV RNA in vitro, inhibit the transcription and expression of F viral gene, reduce the RSV yield by about 7 logs and protect more than 90% RSV-infected Hep-2 cells from a cytopathic effect at 8 μM. Moreover, 10 wild RSV strains isolated from clinic patients including both subgroups A and B were all suppressed by DZn1133 with greater anti-RSV activity than antisense DNA or ribavirin. © 2006 Elsevier B.V. All rights reserved.