Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1(-/-) mice is thin, has a reduced number of cells, is poorly stratified, is depleted of suprabasal wing cells, and its most superficial cell layer blisters. In mature Hmgn1(-/-)mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1(-/-) corneas: glutathione-S-transferase (GST)alpha 4 and GST omega 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and alpha-, beta-, and gamma-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p 63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p 63 expressing cells in the central region of the Hmgn1(-/-) cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.