Using a column-switching HPLC system combining a micro-ODS column and a chiral column, the amounts of D-proline (D-Pro) have been determined in 18 tissues, plasma and urine of mice. To avoid the enzymatic degradation of D-amino acids in vivo, a mutant mouse strain lacking D-amino acid oxidase activity (ddY/DAO(-) mouse) was used. In the brain, relatively large amounts of D-Pro were observed in the anterior pituitary, posterior pituitary and pineal glands. In the peripheral tissues, the amounts of D-Pro were high in the pancreas and kidney. Above all, it is surprising that the ddY/DAO(-) mice excreted large amounts of D-Pro in their urine (433 nmol/mL, 20 times that of L-Pro). The origin of D-Pro has also been investigated. By comparing germ-free mice and gnotobiotic mice, intestinal bacteria were shown to have no effect on the urinary D-Pro amount. Concerning the dietary origin, a notable amount of D-Pro was still excreted in the urine after starvation for 4 days, suggesting that some of the D-Pro is produced in the mice. Age-dependent changes in the urinary D-Pro amount have also been investigated from the postnatal 1st month up to 12 months, and ddY/DAO(-) mice were found to excrete large amounts of D-Pro in the urine constantly throughout their lives.