Inhibition of Na+/K+-ATPase activity from human erythrocyte membranes and commercial porcine cerebral cortex by in vitro single and simultaneous exposure to digoxin and gitoxin was investigated to elucidate the difference in the mechanism of the enzyme inhibition by structurally different cardiac glycosides. The drugs exerted a biphasic dose-dependent inhibitory effect on the enzyme activity in both tissues, supporting the existence of two sensitive Na+/K+-ATPase isoforms. The IC50 values for the low and high affinity isoforms were calculated from the inhibition curves using mathematical analysis. The Hill coefficient (n) fulfilled the relationship 1 < n < 3, suggesting cooperative binding of inhibitors to the enzyme. Kinetic analysis showed that digoxin and gitoxin inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction. Both the isoforms were always more sensitive to gitoxin. The erythrocyte enzyme was more sensitive to the inhibitors in the range of low concentrations but the commercial cerebral cortex enzyme exerted a higher sensitivity in high inhibitors affinity concentration range. By simultaneous exposure of the enzyme to digoxin and gitoxin in combinations a synergistic effect was achieved by low inhibitor concentrations. An antagonistic effect was obtained with erythrocyte membrane enzyme at high inhibitors concentration.