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Introduction: Cardiac diseases can affect the contractile and/or conductive cardiac muscle cells. Recently, the microscopic examination of the subendocardial biopsy is being used in the diagnosis of some cardiac lesions. However, the routine staining cannot differentiate between different types of cardiac fibers. Aim of the work: The present study aims to illustrate the use of anti-cardiac troponin-T3 (cTnT3) immunostaining as a recent tool to distinguish between the contractile and conducting fibers. Materials and Methods: Specimens from both left ventricular wall and interventricular septum were obtained from 6 healthy adult male mice. These specimens were fixed in 4% paraformaldehyde at 4⁰C and processed for paraffin sections. Then, 3 μm thick sections were stained with H&E and anti-cTnT3 antibody and were examined under light microscope. The optical density of the cTnT3 staining affinity of the cardiac fibers was measured morphometrically and the values were statistically analyzed. Results: Examination of H&E stained sections of the left ventricular wall and interventricular septum revealed homogenous staining of all cardiac fibers with an undetectable difference among them. While examination of the immunostained sections with anti-cTnT3 revealed different affinities: the majority of fibers expressed high-positive staining affinity while other fibers had a low or no staining affinity. The high-positive staining affinity was in the form of cytoplasmic transverse dark brownish striations in longitudinally oriented cardiac fibers. The fibers which expressed a low or no staining affinity were either singly scattered especially in the left ventricular wall or grouped in the subendocardial regions of the interventricular septum. Statistically, the optical density values of a low or no staining affinity cardiac fibers were significantly lower (2.5±0.01) than high positive staining affinity cardiac fibers (3.04±0.19). Conclusion: Immunostaining with anti-cTnT3 staining could be added to the routine staining in the examination of cardiac biopsies. [ABSTRACT FROM AUTHOR]