Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.