We have assessed a genetic resistance approach based on antisense RNA to interfere with the prototype Minute Virus of Mice (MVMp), an autonomous parvovirus. MVMp is a cytolytic virus when infecting the permissive A9 mouse fibroblast cell line, and its gene expression is largely regulated at the level of transcription initiation by the nonstructural transactivator NS-1 protein, a multifunctional polypeptide also involved in viral DNA replication and cytotoxicity. An NS-1 specific antisense RNA constitutively expressed in transfected A9 clones increased several fold the proliferative viability of the cells upon high multiplicity virus infection, and cultures infected at low multiplicity reached confluence overcoming virus progression. All clones shared a common phenotype of resistance characterized by a lowered synthesis of viral DNA replicative intermediates and genomic forms, a significant reduction in the accumulation of the three viral messengers in the cytoplasmic and nuclear compartments, and a specific inhibition in viral protein synthesis. These results indicate that the constitutive antisense RNA mediates an overall repression of viral macromolecular synthesis by preventing the onset of NS-1 functions. Therefore, cytocidal parvoviruses may be hampered by engineered antisense RNA targeted against early regulators of virus growth.