Background and Objectives: Fractional photothermolysis is a new concept in cutaneous re-modeling whereby laser-induced microscopic zones of thermal injury (MTZMicroscopic Treatment Zones) are surrounded by normal, viable tissue. This unique thermal damage pattern allows re-epithelialization in less than 24 hours. To increase patient comfort level during the procedure of fractional photothermolysis, simultaneous skin cooling has been proposed and is now extensively used. The purpose of this in vitro study was to examine the influence of skin temperature on the diameter of the epidermal microthermal zone and the extent of thermal injury per unit area. The determination of the changes in these parameters that are due to skin temperature will allow the better control and understanding of fractional photothermolysis at different skin temperatures. Materials and Methods: Fractional photothermolysis was performed with a 1,550 nm fiber laser (Fraxel SR 1 Laser) with 10 mJ per pulse on full-thickness cadaver skin. The skin samples were brought prior to exposure to temperatures that ranged from 0 to 458C. The epidermis of the skin samples was separated by dispase treatment, stained for thermal damage by NBTC stain, and lesion diameter was assessed by a blinded investigator. Results : The average MTZ diameter exhibits a positive, linear relationship with skin temperature (R 2 ¼ 0.904, P < 0.0001). As the skin temperature increases from 0 to 458C. The MTZ diameter increases from 93 to 147 mm (58%), and the MTZ area from 6,870 to 17,050 mm 2 (148%). Conclusion : The skin temperature affects the size of epidermal MTZs during fractional photothermolysis and is an important variable factor. The use of simultaneous skin cooling increases patient comfort; however, as it also decreases MTZ size, it may interfere with treatment efficacy. The control of skin temperature is necessary to provide a consistent outcome and to be able to compare treatments.