The determination of the optimum conditions for the conversion of ergosterol to vitamin D 2 in shitake mushrooms (Lentinula edodes) was studied using response surface methodology (RSM). The effects of the three main variables ambient temperature Holick et al., 2008), which is only found in oily fish such as salmon, sardine, and tuna, as well as in eggs and red meat (Shrapnel and Truswell, 2006). Vitamin D 2 is the form that is generally used in food and pharmaceutical supplementation (Jasinghe and Perera, 2005). Though shitake mushrooms contain a small amount of vitamin D 2 , researchers have found them to be rich in ergosterol (Mattila et al., 2002). In addition, shitake is the most popular mushroom in the eastern Asian countries (Ogra et al., 2004), widely accepted by vegetarians and non-vegetarians. Therefore, shitake mushrooms are chosen as a source of supplemental vitamin D in the present study.Several studies have reported that vitamin D 2 forms in mushrooms that have been exposed to UV irradiation through conversion of ergosterol, pro-vitamin D 2 (Ko et al., 2008;Roberts et al., 2008). Though irradiate factors such as irradiation dose (or intensity), exposure time, ambient temperature and moisture content have been investigated (Jasinghe and Perera, 2005;Jasinghe and Perera, 2006;Ko et al., 2008), the optimum conditions of vitamin D 2 conversion in shitake mushrooms have not been reported. Response surface methodology (RSM) which is a collection of experimental strategies, mathematical methods and statistical inference, is used This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.