OBJECTIVE To explore the possibility of using telomerase as a marker of corneal limbal stem cells. METHODS Corneal limbal tissues and central corneal epithelial tissues from 8 rabbits were examined for telomerase activity qualitatively by telomere repeat amplification protocol (TRAP) and quantitatively by detecting the light value with bioluminescent technique. 5-Fu (20 mg) was injected subconjunctivally in 4 rabbits and the other 4 rabbits were not injected. RESULTS Telomerase activity was positive in all corneal limbal tissues and negative in all central corneal tissues. Telomerase activity of corneal limbal cells (light value 165,575) was significantly higher than that of central corneal epithelial cells (light value 34,912) by bioluminescent technique (P = 0.001). 5-Fu injection group showed higher telomerase activity than that of the group without injection (light value 145,754) (P = 0.02). CONCLUSIONS Positive telomerase activity is detected in the corneal limbal tissues. It suggests that there are cells with high proliferative ability in the corneal limbus. Telomerase activity may be used as a marker of corneal limbal stem cells.