CA IX is a transmembrane carbonic anhydrase isoenzyme predominantly expressed in human tumors in response to hypoxia and functionally implicated in adaptation of tumor cells to hypoxic stress via control of pH and cell adhesion. Intense investigations of the human CA IX as a hypoxic marker and a therapeutic target have been facilitated by specific monoclonal antibodies. However, no such reagents existed for the mouse CA IX ortholog. We generated five new anti-mouse CA IX monoclonal antibodies AM1-4, AM4-3, AM27-4, AM34-7 and AM35-1 produced using CA IX-deficient mice. The antibodies are suitable for various immunodetection methods including immunoblotting and immunohistochemistry. Using these reagents we show that the mouse CA IX is expressed in three out of nine tested mouse cell lines, namely in L929, MEF and TSA and is regulated by hypoxia and cell density similarly to human CA IX. We also demonstrate that the mouse CA IX exhibits hypoxia-related expression pattern in multicellular spheroids and in tumor xenografts. Our results indicate the use of the mouse model as suitable for further studies of CA IX role in tumor development and for its pre-clinical investigations. The new monoclonal antibodies represent potent tools for accomplishment of these future studies.