The expression pattern of tumor necrosis factor-α (TNF-α) mRNA and protein was examined in vivo in experimental mouse skin wounds by in situ hybridization and immunohistochemistry. TNF-α mRNA and protein is detected in a distinct layer of mainly neutrophils subadjacent to the wound clot. The layer of TNF-α-positive cells extends from the margin of the advancing epithelial outgrowth to the opposing one. By in situ hybridization the TNF-α mRNA is detectable 12 h after wounding; the signal peaks after 72 h and remains visible up to at least 120 h after wounding. TNF-α mRNA could not be detected in the normal skin or in 5-hour-old wounds, lmmunohistochemical staining for TNF-α and macrophages on adjacent sections confirms that the main part of TNF-α-positive cells are polymorphonuclear neutrophils and shows that most of the cells located just beneath the layer of TNF-α-positive neutrophils are macrophages with weak TNF-α immunoreactivity. The data reported here show that neutrophils serve as an important source of TNF-α during healing of mouse skin wounds. We suggest that this specific expression of TNF-α is related to the process of re-epithelialization.