To develop an effective strategy to prevent neointima formation after angioplasty injury, we have identified cell-cycle regulatory proteins as targets for inhibition by using antisense oligonucleotides (ODNs). We utilized an intraluminal molecular delivery method that employs the protein coat of a Sendai virus complexed with liposomes that enhances markedly the efficiency of ODNs uptake. First, we examined the effect of antisense cdc2 kinase and proliferating-cell nuclear antigen (PCNA) ODNs in vitro. Cotransfection of antisense cdc2 kinase and PCNA ODNs inhibited serum-stimulated vascular smooth muscle cell growth, whereas antisense cdc2 kinase ODNs alone or PCNA ODNs alone failed to show any inhibitory effect. Transfection of the combination of antisense cdc2 kinase and PCNA ODNs into balloon-injured arteries in vivo provided a marked decrease in cdc2 and PCNA mRNA expression as determined by reverse transcription-PCR, compared to sense controls. Antisense ODN treatment significantly inhibited the increase in DNA synthesis induced by balloon injury. Moreover, antisense ODN administration inhibited completely neointima formation at 2 weeks after angioplasty in an apparent dose-dependent manner. Moreover, the inhibitory effect of antisense ODN on neointima formation persisted up to 8 weeks after a single transfection. The present study documents that a single intraluminal molecular delivery of combined cdc2 kinase and PCNA antisense ODNs results in a sustained inhibition of neointima formation in the rat carotid balloon-injury model.