This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing beta-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3-6.0 and at 40-60 A degrees C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and H-1- and C-13-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other beta-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 mu l of SPGase at pH 4.0 and 55 A degrees C. This is the first report detailing the production of S from ST hydrolysis by a novel beta-glucosidase, which may be useful for the pharmaceutical and agricultural areas.