In this paper, we examined the distribution pattern of D-aspartic acid (D-Asp), as well as D-aspartate oxidase (D-AspO), D-amino acid oxidase (D-AAO), and L-amino acid oxidase (L-AAO) activities in different tissues of frog, Rana esculenta. High concentrations of free D-Asp were found in the testes (0.21+/-0.02 micromol/g b.w), in the liver (0.20+/-0.03 micromol/g b.w), and in the Harderian gland (HG) (0.19+/-0.03 micromol/g b.w). A higher activity of both D-AspO and D-AAO with respect to L-AAO was endogenously present in all examined frog tissues, particularly within the kidney, liver, and brain. Our in vivo experiments, consisting of i.p. injections of 2.0 micromol/g b.w. D-Asp in frogs, revealed that all examined tissues can take up and accumulate D-Asp and that this amino acid specifically triggers D-AspO activity. Indeed, no increase in both D-AAO and L-AAO was found in all frog tissues after D-Asp treatment. The optimum pH for D-AspO activity was around 8.2 and the optimum temperature was about 37 degrees C. Furthermore, its activity linearly increased with increasing D-Asp incubation times. In vitro experiments assaying the substrate specificity of D-AspO indicated that the enzyme had greater affinity for N-methyl-D-aspartate than for D-Asp and D-glutamate. This study provides evidence of the presence of free D-Asp in frog R. esculenta tissues, along with its role in triggering D-AspO activity. These findings suggest that D-AspO could play an essential role in decreasing excessive amounts of D-Asp in frog tissues, a phenomenon that, if left unchecked, could have detrimental physiological effects on the animal.