The causes of the resistance of adult rats to cardiac glycosides were examined using two approaches: (a) alterations in the binding parameters of ouabain to post-natal heart cells during their maturation in culture, a process which could be indicative of aging; and (b) changes in the I 50 values of cardiac (Na + + K +)-ATPase and K +-phosphatase activities by ouabain during the process of enzyme's purification, from post-natal to adult rats, which could be indicative of structural changes. The amounts of the cell-bound ouabain decreased from 120 to 12 fmol/mg protein or from 8 to 2 fmol/μg DNA between the 2nd and the 10th day or between the 2nd and the 8th day of growth, respectively. Furthermore, partially purified preparations of (Na + + K +)-ATPase from post-natal rat hearts, obtained following treatment with deoxycholate and high concentrations of NaI, were most sensitive to inhibition by ouabain ( I 50 = (4.6±0.6) · 10 −5 M) than similar preparations from adult rats ( I 50 = (22.4±5.3) · 10 −5 M, P<0.005). The I 50 values for the detergent-treated enzyme and the salt-treated enzyme in the adult rat or adult cat hearts were: (22.4±5.3)·10 −5 M and (4.89±0.83)·10 −5 M ( P<0.001), respectively, for the rat and (178±52)·10 −8 M and (2.0±0.48)·10 −8 M ( P<0.005), respectively, for the cat. Frozen and thawed homogenates of either species responded neither to the cations nor to inhibition by ouabain. The K +-activated, ouabain-inhibited changes in p-nitrophenylphosphatase sensitivity to inhibition by the cardiac glycoside were minimal during the purification of the enzyme from both the cat or rat hearts, although the purification factors were 6.4 and 9.0, respectively. The decrease in ouabain binding to new-born rat cardiac muscle cells, but not to non-muscle cells, during maturation in culture; the decrease in sensitivity of (Na + + K +)-ATPase from new-born rats, compared to adult rats, to inhibition by ouabain; and the increase in the sensitivity of the enzyme to inhibition by the drug during the process of the enzyme's purification from adult rat or from cat suggested that certain structural alterations could occur in the ouabain receptor during aging in culture or maturation in vivo. These may be reflected by the removal of certain components during the process of the enzyme's purification and may provide a partial explanation for the resistance of the rat to ouabain.