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Nattokinase is a potent fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. The enzymes were discovered from various sources, such as Japanese natto, Korean Chungkookjang soy sauce, and Chinese douchi. Intravenous fibrinolytic agents such as urokinase and tissue plasminogen activator have been widely used in clinical practice for thrombolytic therapy. Microbial fibrinolytic enzymes have now attracted much more attention than typical thrombolytic agents because of the expensive prices and the undesirable side effects of the latter.In this study, Bacillus subtilis was cultivated to high cell density for nattokinase production. Effect of supplementing complex media (peptone, yeast extract, or tryptone) was investigated on cell growth and nattokinase production. Among complex media, the highest cell growth (optical density monitored at 600 nm) and nattokinase activity were obtained with peptone supplementation. Amino acid analysis of the three complex media showed that glycine was richest in peptone. In fed-batch culture of B. subtilis, optimum ratio of peptone to glucose in feeding solution was 3:1. At this condition, cell concentration and nattokinase activity were 75 g/L and 15,000 unit/ml, respectively. By supplementing 2 g/L glycine in peptone-containing media, nattokinase activities increased compared without glycine supplementation