We have identified and quantified free D-aspartate in adult rat peripheral organs using gas chromatographic-mass spectrometric and high-performance liquid chromatographic techniques. The level of free D-aspartate was highest in the adrenal, testis, spleen and pituitary, followed by the thymus, lung, ovary, placenta, pancreas and heart, and below the detection limit in the kidney, liver, brain, muscle and serum. These data provide the first evidence that a high level of free D-aspartate widely occurs in the adult rat periphery and suggest that the D-amino acid may be an endogenous substrate for D-aspartate oxidase.