Contents and sources of glycerol in barley shochu mash (BSM) were investigated. Glycerol released by lipase (T-Gly) was approximately 4.1mg/g dried barley, and 9.3mg/g dried barley-koji, respectively. The T-Gly concentration was influenced by the activity of lipase, and higher in barley than in barley koji. Glycerol in barley koji consisted of the glycerol formed during koji making (F-Gly) and the other newly released glycerol by lipase (G-Gly). The amount of F-Gly was less than that of G-Gly until 30 h of koji making, but the amount of F-Gly increased steeply after 36 h. Glycerol in BSM was varied according to koji ratio (% of koji weight to the total materials weight). The concentration of glycerol observed was 6.1, 7.8 and 10. 1g/l for koji ratios 33%, 60% and 100%, respectively. It is inferred that glycerol formation by yeast at koji ratio 100% was increased approximately by 25%(w/w) than that obtained at koji ratio of 33%.