NG-nitro-D-arginine (D-NNA) produced pressor responses in rats by acting via chiral inversion into NG-nitro-L-arginine (L-NNA), an inhibitor of nitro oxide synthase. The present investigation aimed to study the role of the D-amino acid oxidase (DAAO) in chiral inversion of D-NNA and the relationship between DAAO activities on various D-amino acids and their inversion rate. Benzoate (400 mg/kg) or creatinine (400 mg/kg), two inhibitors of DAAO, blocked D-NNA-induced pressor responses in rats. Furthermore, the addition of the pure DAAO significantly potentiates L-NNA production rate in kidney homogenates by approximately 2-folds. The in vivo and in vitro results suggested that DAAO plays an essential role in the pressor responses elicited by D-NNA. Moreover, crude DAAO solution from the kidney showed marked selection (the maximal ratio of Kcat/Km was nearly 15 times) on different D-amino acids that exhibited similar chiral inversion rate in vivo, suggesting that other enzymes, such as transaminase, are also required for the entire process of D-NNA chiral inversion.