Myocardial Na+,K+-ATPase was studied in patients with aortic valve disease, and myocardial Na+,K+- and Ca2+-ATPase were assessed in spontaneously hypertensive rats (SHR) and hereditary cardiomyopathic hamsters using methods ensuring high enzyme recovery. Na+,K+-ATPase was quantified by [3H]ouabain binding to intact myocardial biopsies from patients with aortic valve disease. Aortic stenosis, regurgitation and a combination hereof were compared with normal human heart and were associated with reductions of left ventricular [3H]ouabain binding site concentration (pmol/g wet weight) of 56, 46 and 60%, respectively (p < 0.01). Na+,K+- and Ca2+-ATPases were quantified by K+- and Ca2+-dependent p-nitrophenyl phosphatase (pNPPase) activity determinations in crude myocardial homogenates from SHR and hereditary cardiomyopathic hamsters. When SHR were compared to age-matched Wistar Kyoto (WKY) rats an increase in heart-body weight ratio of 75% (p < 0.001) was associated with reductions of K+- and Ca2+-dependent pNPPase activities (μmol/min/g wet weight) of 42 (p < 0.01) and 27% (p < 0.05), respectively. When hereditary cardiomyopathic hamsters were compared to age-matched Syrian hamsters an increase in heart-body weight ratio of 69% (p < 0.001) was found to be associated with reductions in K+- and Ca2+-dependent pNPPase activities of 50 (p < 0.001) and 26% (p = 0.05), respectively. The reductions in Na+,K+- and Ca2+-ATPases were selective in relation to overall protein content and were not merely the outcome of increased myocardial mass relative to Na+,K+- and Ca2+-pumps. In conclusion, myocardial hypertrophy is in patients associated with reduced Na+,K+-ATPase concentration and in rodents with reduced Na+,K+- and Ca2+-ATPase concentrations. This may be of importance for development of heart failure and arrhythmia in hypertrophic heart disease.