From the cow milk casein digests with alkaline proteinase of Bacillus subtilis was isolated a bitter peptide consisting of only tryptophan and leucine which was soluble in chloroform, but sparingly soluble in water. The peptide was attacked neither by aminopeptidase nor carboxypeplidase and showed no electrophoretic mobility. The peptide was negative in detection tests for amino and carboxyl groups. A partial hydrolysis by acid resulted in loss of bitterness and the hydrolysates became positive in the tests for amino and carboxyl groups. Amino acid analysis revealed that the peptide was composed of equimolar tryptophan and leucine, and a cyclic structure consisting of two moles of tryptophylleucine was suggested for the bitter peptide.