A method for the separatory determinaton of bacterial α-amylase in a mixture of pancreatic, Aspergillus and bacterial α-amylase was investigated. Treatment with 8M urea was used for this separatory determination. After preincubation with 8M urea at 37° for 30 min, 83.4% of activity of bacterial α-amylase was recovered by dilution while the other α-amylases were almost denatured completely. This property of bacterial α-amylase was applied to the separatory determination. Activity of bacterial α-amylase and total activity of mixed amylases were obtained in the presence or absence of 8M urea, respectively. The average percentage of recovery of bacterial α-amylase was 100.8±7.6%. Various compounds, except sodium chloride, used in drugs did not affect the practical application of this method.