Agaricus blazei Murrill, an edible mushroom, is widely used as a functional food due to its possible medicinal effects. Aqueous extracts are also used as food additive to provide an agreeable bitter taste. As a part of its safety assessment, the present 90-day subchronic toxicity study was performed in F344 rats. To establish a no-observed-adverse-effect level (NOAEL), rats were fed powder diet containing A. blazei Murrill aqueous extract at dose levels of 0 (basal diet), 0.63, 1.25, 2.5 and 5% (maximum) for 90 days. During the experiment, there were no remarkable changes in general appearance and no deaths occurred in any experimental group. Although serum blood urea nitrogen was slightly but significantly increased in males of the 2.5 and 5% groups, no related histopathological changes were observed in the kidney, and serum creatinine levels were rather reduced, suggesting the increase of blood urea nitrogen to be of little toxicological significance. Hematology, organ weight measurement and histopathological observation revealed no test compound-related toxicological changes. In conclusion, A. blazei Murrill extract even at 5% in the diet (2654 mg/kgb.w./day for male rats and 2965 mg/kgb.w./day for female rats) did not cause remarkable adverse effects in F344 rats. Thus, the NOAEL was concluded to be 5% in the diet.