The occurrence of free D-aspartic acid was measured in various tissues and organs of the octopus Octopus vulgaris by means of chiral HPLC amino acid analysis. This amino acid was detected in appreciable quantities in almost all of the tissues examined (muscle, renal appendage, ventricle, branchial heart, gill, crop, stomach, cecum, intestine, salivary gland, brain, and eye) except the hepatopancreas and gonad. In particular, the amount of D-aspartic acid in the intestine was 5- to 10-fold that in other tissues. The ratios of D/L were 2 or more in brain, eye, and intestine, but were less than 1 in the other tissues. D-Aspartate oxidase (EC 1.4.3.1) activity was found in various digestive organs and in the muscle of the octopus, but it was not detected in the brain, eye, gill, salivary gland, or gonad. The enzyme activity was highest in the cecum (1.3U/g tissue) and was relatively high in the renal appendage, hepatopancreas, and intestine (0.8, 0.6 and 0.5U/g tissue, respectively). The oxidizing activity towards L-aspartic acid was only slightly detected in all of the tissues examined. These results imply that the D-aspartic acid in octopus tissues originates mainly from intestinal bacteria and feed, and that the D-aspartate oxidase secreted from the cecum controls the accumulation of this amino acid.