Purpose To assess the suitability of a new 345 nm ultraviolet (UV) femtosecond laser for refractive surgery. Setting Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany. Design Experimental study. Methods Twenty-five porcine corneas were used for stromal flap or lamellar bed creation (stromal depth, 150 μm) and 15 rabbit corneas for lamellar bed creation near the endothelium. Ultraviolet femtosecond laser cutting-line morphology, gas formation, and keratocyte death rate were evaluated using light and electron microscopy and compared with a standard infrared (IR) femtosecond laser. Endothelial cell survival was examined after application of a laser cut near the endothelium. Results Flaps created by the UV laser were lifted easily. Gas formation was reduced 4.2-fold compared with the IR laser (P =.001). The keratocyte death rate near the interface was almost doubled; however, the death zone was confined to a region within 38 μm ± 10 (SD) along the cutting line. Histologically and ultrastructurally, a distinct and continuous cutting line was not found after UV femtosecond laser application if flap lifting was omitted and standard energy parameters were used. Instead, a regular pattern of vertical striations, presumably representing self-focusing induced regions of optical tissue breakdown, were identified. Lamellar bed creation with standard energy parameters 50 μm from the endothelium rendered the endothelial cells intact and viable. Conclusion The new 345 nm femtosecond laser is a candidate for pending in vivo trials and future high-precision flap creation, intrastromal lenticule extraction, and ultrathin Descemet-stripping endothelial keratoplasty. Financial Disclosures Mr. Klenke and Ms. Skerl were paid employees of Wavelight GmbH when the study was performed. Dr. Seiler is a scientific consultant to Wavelight GmbH. No other author has a financial or proprietary interest in any material or method mentioned.