BACKGROUND The mechanical stabilization of the cornea in keratoconus may delay progression of this disease. The cross-linking techniques optimized in corneas of enucleated porcine eyes were investigated under in vivo conditions in rabbits to estimate the biocompatibility and duration of the stiffening effect. METHODS Twenty-eight rabbits were treated monocularly, the fellow eye serving as control. The epithelium was mechanically removed and 19 eyes were treated with riboflavin plus ultraviolet irradiation (365 nm, 2 mW/cm2) for 45 min and 9 eyes with 0.075% glutaraldehyde for 20 min. After treatment, the eyelids were sutured for 3 days. The healing process was controlled by slit-lamp examination and photographically documented. After 1 month, 20 animals and after 3 months 8 animals were sacrificed, the eyes enucleated, and the stress-strain relation of the corneas measured and compared to the fellow eye. RESULTS The epithelium was closed after 4-5 days. The transparency of the corneas remained clear during follow-up, and there were no signs of inflammatory reaction. Stress for a strain of 6% was higher in the treated corneas by a factor of 1.3 +/- 0.66 (P = 0.319) in the glutaraldehyde group and by a factor of 1.6 +/- 0.75 (P = 0.0408) in the riboflavin group at 1 month, and by 1.3 +/- 0.48 (P = 0.07) at 3 months after treatment. CONCLUSIONS The cross-linking technique using riboflavin plus UV irradiation is suitable for at least temporarily stiffening the cornea in vivo and seems to be a promising method for conservative treatment of keratectasia.