Using novel nitric oxide (NO)-generating polymeric hydrogels that can be rapidly photopolymerized in situ, we can deliver NO locally at the site of vascular injury. Depending on material design, these poly(ethylene glycol) (PEG)-based hydrogels can generate NO for up to 50 d. This study demonstrates the ability of nitric oxide-generating hydrogels (PEG-Cys-NO) to influence key components of the restenosis cascade both in vitro and in vivo. PEG-Cys-NO hydrogels inhibited smooth muscle cell proliferation, increased endothelial cell proliferation, and inhibited platelet adhesion in vitro. Moreover, in vivo, PEG-Cys-NO hydrogels inhibited intimal thickening in a rat carotid balloon injury model. The perivascular application of NO-generating polymers post-injury reduced neointima formation at 14 d by approximately 80% compared to controls (intimal area/medial area (I/M): PEG-Cys-NO=0.20+/-0.17, control=0.84+/-0.19, p<0.00002; intimal thickness: PEG-Cys-NO=12+/-10 microm, control=60+/-18 microm, p<0.00002). Treatment with the PEG-Cys-NO hydrogels caused a significant decrease in the per cent of proliferating cell nuclear antigen positive medial cells (29+/-5%) at 4 d as compared to treatment with the control hydrogels (51+/-1%, p<0.02). Additionally, vessel re-endothelialization at 14 d was slightly enhanced in the presence of the NO-generating hydrogels. These data indicate that localized delivery of NO from these hydrogels can significantly inhibit neointima formation in a rat carotid balloon injury model and suggest that these materials may be useful in preventing restenosis.