AIM:To evaluate the enrichment of riboflavin in the corneal stroma after intracameral injection to research the barrier ability of the corneal endothelium to riboflavin in vivo.METHODS:The right eyes of 30 New Zealand white rabbits were divided into three groups. Different concentrations riboflavin-balanced salt solutions (BSS) were injected into the anterior chamber (10 with 0.5%, 10 with 1%, and 10 with 2%). Eight corneal buttons of 8.5 mm in diameter from each group were dissected at 30min after injection and the riboflavin concentrations in the corneal stroma were determined using high-performance liquid chromatography (HPLC) after removing the epithelium and endothelium. The other two rabbits in every group were observed for 24h and sacrificed. As a comparison, the riboflavin concentrations from 16 corneal stromal samples were determined using HPLC after instillation of 0.1% riboflavin-BSS solution for 30min on the corneal surface (8 without epithelium and 8 with intact epithelium).RESULTS: The mean riboflavin concentrations were 11.19, 18.97, 25.08, 20.18, and 1.13 μg/g for 0.5%, 1%, 2%, de-epithelialzed samples, and the transepithelial groups, respectively. The color change of the corneal stroma and the HPLC results showed that enrichment with riboflavin similar to classical de-epithelialized corneal collagen crosslinking (CXL) could be achieved by intracameral1% riboflavin-BSS solution after 30min; the effect appeared to be continuous for at least 30min.CONCLUSION:Riboflavin can effectively penetrate the corneal stroma through the endothelium after an intracameral injection in vivo, so it could be an enhancing method that could improve the corneal riboflavin concentration in transepithelial CXL.