Purpose: The aim of this study was to investigate the possibility of delivering riboflavin into the corneal stroma for cross-linking with the aid of a tensioactive substance (benzalkonium chloride), without removing the epithelium, in order to make corneal cross-linking (CXL) a less invasive technique. Methods: Rabbit corneas were cross-linked in vivo without removal of the epithelium, using riboflavin solution 0.1% to which benzalkonium chloride solution was added. The corneas were then examined by light and electron microscopy for evidence of collagen cross-linking. Control non-treated eyes were compared with treated ones. Results: The treated corneas were stiff and yellow in colour, and on light microscope examination they showed straight compact collagen fibres in comparison with control corneas in approximately 50% of the corneal thickness. Electron microscopy revealed regular arrangement of fibres in the outer 200µm with reduced inter-fibre distances in the treated corneas, but with no noticeable differences between control and treated corneas in the inner 100µm. Conclusion: The addition of benzalkonium chloride to the riboflavin solution facilitated its entry to the corneal stroma through the epithelium, and CXL occurred without epithelium removal. KeywordsBenzalkonium chloride, collagen fibres, corneal cross-linking, epithelium, riboflavin, ultraviolet A Disclosure: The authors have no conflicts of interest to declare. Materials and MethodsThe cross-linking was carried out at the Zooprophylaxis Institute,