Mutations in the winged-helix nude (whn) gene result in the nude mouse and rat phenotypes. The pleiotropic nude phenotype which affects the hair, skin, and thymus suggests that whn plays a pivotal role in the development and/or maintenance of these organs. However, little is known about whn function in these organs. We show here that in skin whn is specifically expressed in epithelial cells and not the mesenchymal cells, and using a hair reconstitution assay, we demonstrate that the abnormal nude mouse hair development is attributable to a functional defect of the epithelial cells. Examination of nude mouse primary keratinocytes in culture revealed that these cells have an increased propensity to differentiate in an abnormal fashion, even under conditions that promote proliferation. Furthermore, nude mouse keratinocytes displayed a 100-fold increased sensitivity to the growth-inhibitory/differentiation effects of the phorbol ester TPA. In parallel with these findings, we directly show that whn functions as a transcription factor that can specifically suppress expression of differentiation/TPA-responsive genes. The region of Whn responsible for these effects was mapped to the carboxy-terminal transactivating domain. These results establish whn as a key regulatory factor involved in maintaining the balance between keratinocyte growth and differentiation. The general implications of these findings for an epithelial self-renewal model will be discussed.