Recently, we demonstrated a large induction of inducible nitric oxide synthase (iNOS) during cutaneous wound repair. In this study, we established an in vivo model in mice to investigate the role of NO during the wound healing process. During excisional repair, mice were treated with L-N6-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS enzymatic activity. Compared with control mice, L-NIL-treated animals were characterized by a severely impaired reepithelialization process, as the hyperproliferative epithelia at the wound edges appeared to be delayed and characterized by an atrophied morphology. Immunohistochemical labeling for detection of proliferating cells (BrdU-, Ki67-staining) revealed a strong reduction in proliferating keratinocyte cell numbers during the process of re-epithelialization after inhibition of iNOS activity during repair. Western blot analysis of total wound lysates from PBS- and L-NIL-treated mice clearly demonstrated a reduction in proliferating cell nuclear antigen, representing a marker for cell proliferation, in lysates isolated from L-NIL-treated mice. The dependency between keratinocyte proliferation and NO availability observed during wound repair in vivo is further supported by the observation that proliferation of the keratinocyte cell line (HaCaT) is stimulated by low concentrations of NO-donors also in vitro. In summary, our data demonstrate that the presence of a functionally active iNOS is a crucial prerequisite for normal wound reepithelialization.