Na,K-ATPase concentration was measured by vanadata facilitated3H-ouabain binding to intact samples taken from various parts of porcine and canine myocardium. In porcine and canine heart3H-ouabain binding site concentration in ventricles was 1.4-2.5 times larger than in atria. Evaluation of3H-ouabain binding kinetics revealed no major difference between atria and ventricles: Equilibrium was obtained after the same incubation time in right atrium (RA) as in left ventricle (LV), both in porcine and canine heart. Unspecific uptake and retention of3H-ouabain was for porcine heart RA and LV 1.5 and 1.4, respectively, and for canine heart RA and LV, both 1.2% filling (i.c., volume (ml) of incubation medium3H-radioactivity taken up per mass unit (g wet wt.) of tissue multiplied by 100). The apparent dissociation constant (Kd) was 1.4×10-8 and 1.9×10-8 in porcine RA and LV and 2.6×10-8 and 6.1×10-8 mol/l in canine RA and LV. Loss of specifically bound3H-ouabain during the washout procedure occurred with a half-life time (T1/2) of 16.7 in RA and LV of porcine heart and 91.2 and 151.6h in RA and LV of canine heart. Duly corrected for these errors of the method-factor 1.16 and 1.13, respectively, for porcine RA and LV, and factor 1.11 and 1.13 for canine RA and LV, total3H-ouabain binding site concentration was found to be 553±74 and 1037±45 pmol/g wet wt. (means±SEM, n=5) in porcine RA and LV, and 569±37 and 1410±40 pmol/g wet wt. (means ±SEM, n=5) in the canine RA and LV. These values were confirmed by measurements of3H-digoxin binding to the porcine heart. The present quantification of myocardial Na, K-ATPase gives values up to 154 times higher than measurements based upon Na,K-ATPase activities in membrane fractions where the recovery of Na,KK-ATPase may be less than 1% due to loss during purification. A higher Na,K-ATPase concentration is found in small animals than in large animals. A relationship between higher concentration of Na, K-ATPase and larger pressure work in ventricles compared to atria is suggested. Myocardial3H-ouabain binding sites were found to be stable for 20 min of ischemia, followed by 1h of reperfusion, supporting the concept that myocyte injury induced by short term ischemia may be reversible and that reperfusion may result in normalization.