Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that oxidative stress can regulate extracellular matrix in cardiac fibroblasts. Neonatal and adult rat cardiac fibroblasts in vitro were exposed to H₂O₂ (0.05–5 μM) or the superoxide-generating system xanthine (500 μM) plus xanthine oxidase (0.001–0.1 mU/ml) (XXO) for 24 h. In-gel zymography demonstrated that H₂O₂ and XXO each increased gelatinase activity corresponding to matrix metalloproteinases (MMP) MMP-13, MMP-2, and MMP-9. H₂O₂ and XXO decreased collagen synthesis (collagenase-sensitive [³H]proline incorporation) without affecting total protein synthesis ([³H]leucine incorporation). H₂O₂ and XXO decreased the expression of procollagen α₁(I), α₂(I), and α₁(III) mRNA but increased the expression of fibronectin mRNA, suggesting a selective transcriptional effect on collagen synthesis. H₂O₂, but not XXO, also decreased the expression of nonfibrillar procollagen α₁(IV) and α₂(IV) mRNA. To determine the role of endogenous antioxidant systems, cells were treated with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DDC, 100 μM) to increase intracellular superoxide or with the glucose-6-phosphate dehydrogenase inhibitor dehydroisoandrosterone 3-acetate (DHEA; 10 μM) to increase intracellular H₂O₂. DDC and DHEA decreased collagen synthesis and increased MMP activity, and both effects were inhibited by an SOD/catalase mimetic. Thus increased oxidative stress activates MMPs and decreases fibrillar collagen synthesis in cardiac fibroblasts. Oxidative stress may play a role in the pathogenesis of myocardial remodeling by regulating the quantity and quality of extracellular matrix.