Real-time PCR was optimized for the quantification of canine CD56 mRNA expression. This study was conducted to easily quantify canine CD56 expression and to identify its expression in normal tissues, peripheral blood mononuclear cells and activated lymphocytes in dogs. This assay revealed the highest level of CD56 mRNA expression in the normal canine brain, followed by the lung, kidney and liver. CD56 mRNA expression level in peripheral blood mononuclear cells was considerably lower; among activated lymphocytes in vitro, CD56 mRNA expression was increased.