Previous investigations demonstrated that fibronectin is an essential adhesive glycoprotein for the corneal epithelial cells. Fibronectin mediates attachment and spreading of the corneal epithelial cells. However, it seems to be important to know the changes in cellular receptor activity in order to understand the interactions of corneal epithelial cells and underlying extracellular matrix. In this investigation, we studied the effects of various culture times and conditions on the receptor activity of rabbit corneal epithelial cells for fibronectin. The cultured cells were placed on tissue culture plates that were coated with various concentrations of four extracellular matrices: fibronectin, laminin and collagen types I and IV. Freshly isolated corneal epithelial cells without culture did not spread on fibronectin and laminin; they spread only on collagen types I and IV. When the epithelial cells were cultured for 12 h or more, they spread on fibronectin. However, the spreading of the cells on collagen types I and IV was the same regardless of the culture period (up to 20 h). Only a small number of epithelial cells spread on laminin at the highest concentration examined after culture for 12 h or more. Thus, the corneal epithelial cells responded differently to fibronectin, collagen types I and IV, and laminin. Perhaps the receptor for fibronectin appears when epithelial cells are cultured, but the receptor(s) for collagen types I and IV are always present on the cell surface of corneal epithelial cells.