OBJECTIVE To use nanoscopically defined, two-dimensional matrices assembled from aligned collagen type I fibrils as a sheet substratum for in vitro cultivation of human corneal endothelial cells (HCECs). To assess the effect of matrix architecture on HCEC morphology and to characterize integrin-mediated HCEC-matrix interaction. METHODS Cell alignment and cell-matrix interactions of primary HCECs and three different immortalized HCEC populations on native and UV-cross-linked collagen type I matrices were examined by time-lapse microscopy. Specific integrin α(2)β(1) binding to the collagen matrix was demonstrated using a function-blocking α(2) antibody. Integrin α(2) subunit expression levels of the four HCEC populations were analyzed by Western blot analysis. RESULTS All HCEC populations aligned along the oriented collagen fibrils. Primary HCECs and, to a lesser extent, the other tested HCEC populations exerted high traction forces, leading to progressive matrix destruction. Cross-linking of the collagen matrices considerably increased matrix stability. Integrin subunit α(2) expression levels of the four cell types correlated with the degree of cell alignment and exertion of traction forces. In turn, blocking integrin subunit α(2) reduced cell alignment and prevented matrix destruction. CONCLUSIONS HCECs align directionally along parallel arrays of collagen type I fibrils. The interactions of HCECs with collagen type I are primarily mediated by integrin α(2)β(1). Integrin subunit α(2) levels correlate with matrix contraction and subsequent destruction. Sustained cultivation of HCECs on ultrathin collagen matrices thus requires matrix cross-linking and moderate integrin α(2)β(1) expression levels.