The total Na+-K+-ATPase activity in rodent heart ventricle was quantified by determination of K+-dependent, ouabain suppressible 3-O-methylfluorescein phosphatase activity. A K+-dependent phosphatase activity of 0.80 μmol/min per g wet wt was obtained from crude homogenate of heart ventricle from 4-week-old guinea pigs. The anticipated association between K+-dependent phosphatase activity and Na+-K+-activated ATP hydrolysis could be clearly demonstrated in the crude homogenate. Based upon a molecular activity of 550/min the corresponding cardiac glycoside receptor concentration in the crude homogenate was 1450 pmol/g wet wt. In crude homogenate of heart ventricle from 3-month-old rats the K+-dependent phosphatase activity was 1.16 μmol/min per g wet wt. Following 4 weeks of K+ depletion of the rats, a decrease of 13% in total K+ content of the heart ventricle was seen. This was associated with a 14% decrease in K+-dependent phosphatase activity. The induction of hypo- and hyperthyroidism for 3 weeks in rats was followed by a decrease of 27% and an increase of 13% in K+-dependent phosphatase activity, respectively.