PURPOSE To determine the effect of the duration of irradiation on the biomechanical parameters of combined riboflavin-ultraviolet-A (UVA) collagen crosslinking (CXL) in rabbit sclera. SETTING Department of Ophthalmology, Provincial Hospital affiliated with Shandong University, Shandong, China. DESIGN Experimental study. METHODS Thirty-six New Zealand rabbits were divided into 6 groups based on the duration of irradiation (10, 20, 30, 40, 50, or 60 minutes). After the application of riboflavin 0.1% drops (without dextran) as a photosensitizer, the animals were irradiated with 3 mW/cm(2) UVA at 365 nm. Only the left eye of each rabbit was treated. All the animals were humanely killed 24 hours postoperatively. One eye in each treated group was used for light microscopy. The other treated eye and all control eyes were prepared for biomechanical testing. The biomechanical parameters were ultimate stress, Young modulus, and the physiological modulus. RESULTS The eyes irradiated for 10 or 20 minutes did not differ significantly from the control eyes. Stress-strain measurement of scleral strips irradiated for 40 minutes or longer showed a significant increase in the ultimate stress, Young modulus, and the physiological modulus. There was a significant increase in the physiological modulus of scleral strips irradiated for 30 minutes or longer. Eyes that were irradiated for 50 minutes and 60 minutes had retinal damage. CONCLUSIONS Riboflavin-UVA CXL can lead to a noticeable increase in the biomechanical stiffness of the sclera. The physiological modulus is the most sensitive tool to measure stiffness. In this study, the optimum duration of irradiation was 40 minutes.