Animal models are important for the investigation of human heart pathology, novel treatments, and medical or surgical interventions for disease. Serum markers of myocardial damage may also be important tools within this field of research. In order to assess the cardiac specificity of widely utilised serum markers, we measured the cardiac troponins and creatine kinase (CK) isoenzymes in cardiac and skeletal muscle samples taken from dog, monkey, pig and rat. These samples were also analysed by immunoblotting for cardiac troponin I (cTnI) and cardiac troponin T (cTnT). The content of cTnI and cTnT in skeletal muscle was below 0.6% of that found in heart for all animal species studied. This low immunoreactivity in skeletal muscle was confirmed by immunoblot analysis. The content of CK was higher in skeletal muscle than in heart muscle for all species. The CK-MB/total CK ratio was lower in skeletal muscle than in cardiac muscle for all species. The differences in CK-MB content of skeletal muscle and heart muscle were much less pronounced than the tissue differences in the amounts of the cardiac troponins. The cardiac troponins are potentially useful serum markers of myocardial damage, with high specificity for myocardial muscle in these common laboratory animals. Creatine kinase-MB is much less cardiac-specific.