To gain more insight into the mechanistic processes controlling the kinetics of inotropic response of digoxin in the perfused whole heart, an integrated kinetic model was developed incorporating digoxin uptake, receptor binding (Na⁺/K⁺-ATPase inhibition) and cellular events linking receptor occupation and response. The model was applied to data obtained in the single-pass Langendorff perfused rat heart for external calcium concentrations of 0.5 and 1.5 mM under control conditions and in the presence of the reverse mode Na⁺/Ca²⁺ exchange inhibitor KB-R7943 (0.1 µM) in perfusate. Outflow concentration and left ventricular developed pressure data measured for three consecutive doses (15, 30, and 45 µg) in each heart were analyzed simultaneously. While disposition kinetics of digoxin was determined by interaction with a heterogeneous receptor population consisting of a high affinity/low capacity- and a low affinity/high capacity-binding site, response generation was more than 80% mediated by binding to the high affinity receptor. Digoxin sensitivity increased at lower extracellular calcium concentrations due to higher stimulus amplification. Coadministration of KB-R7943 significantly reduced the positive inotropic effect of digoxin at higher doses (30 and 45 µg) and led to a saturated and delayed receptor occupancy-response relationship in the cellular effectuation model. The results provide further evidence for the functional heterogeneity of the Na⁺/K⁺-ATPase and suggest that in the presence of KB-R7943 a reduction of the Ca²⁺ influx rate via the reverse mode Na⁺/Ca²⁺ exchanger might become the limiting factor in digoxin response generation.