Recently, there has been renewed interest in the role of reactive oxygen species (ROS), especially H₂O₂, in wound healing. We previously showed that H₂O₂ stimulates healing in a keratinocyte scratch wound model. In this paper, we used a more complex and physiologically relevant model that involves co-culturing primary keratinocytes and fibroblasts. We found that the two main cell types within the skin have different sensitivities to H₂O₂ and to the widely used “antioxidant”N-acetyl-l-cysteine (NAC). Keratinocytes were very resistant to the toxicity of H₂O₂ (250 and 500μM) or NAC (5mM). However, the viability of fibroblasts was decreased by both compounds. Using the co-culture model, we also found that H₂O₂ increases re-epithelialization while NAC retards it. Our data further illustrate the possible role of ROS in wound healing and the co-culture model should be useful for screening agents that may influence the wound healing process.