The behavior of salicylic acid-D-alanine conjugate (salicyl-D-alanine) following intravenous, oral and intracecal administration was examined in rabbits, then compared with that of salicylic acid-L-alanine conjugate (salicyl-L-alanine) as reported previously. Following intravenous administration, salicyl-D-alanine eliminated rapidly from the blood, and its blood concentration was almost identical with that of salicyl-L-alanine. In both cases, salicylic acid could not be detected in the blood, indicating that systemic de-conjugation of D-alanine might not occur. Unchanged salicyl-D-alanine was found in the blood mainly following oral and intracecal administration of salicyl-D-alanine. On the other hand, salicylic acid formed extensively following oral and intracecal administration of salicyl-L-alanine, suggesting that the presystemic de-conjugation of D-alanine and L-alanine was unequal. Furthermore, in vitro incubation of salicyl-D-alanine with cecal content, in which the major source of salicyl-L-alanine hydrolysis is found, showed that the hydrolysis of salicyl-D-alanine was negligible in rabbit intestinal microorganisms.