The concentrations of hyaluronan (HA) and chondroitin sulfate (CS) in rabbit tears were determined using high performance liquid chromatography. The tears were taken from both normal eyes and eyes with a corneal epithelial defect. The concentration of CS was 7.61 times higher than that of HA in the tears of normal eyes. When the concentration of CS isomers was compared, the highest concentration was observed in chondroitin 6-sulfate (C6S), followed by chondroitin 4-sulfate (C4S) and chondroitin (C0S), in that order. When the corneal epithelium was removed from the left eye using a trephine and microscissors, a significant increase was detected in the concentrations of HA and C6S in tears of the left eye one day after removal, whereas no change was observed in those of the right eye. In addition, a significant positive correlation was observed between the increased amount of HA concentration and the healing rate of the corneal epithelium. These results suggest that the increase of HA concentration in tears is associated with spontaneous corneal epithelial healing.