Binding of cardiac glycosides to the Na+,K+-dependent ATPase has been shown to occur at both high- and low-affinity sites. However, recent reports suggest that glycoside-induced inhibition of electrogenic Na-K pump current occurs with simple first-order binding kinetics at relatively low-affinity sites. This implies that high-affinity binding sites have little to do with Na-K pump inhibition during exposure to cardiac glycosides. To better understand the role of the high-affinity site, we investigated the concentration dependence of Ipumpinhibition by dihydroouabain (DHO) hi guinea pig ventricular myocytes through use of wide-pore patch pipettes to "fix" internal Na+activity at ˜30 mM and to voltage clamp at -40 mV (T=34± C). DHO was found to have no effect on membrane conductance at a holding potential of -40 mV. Holding current was monitored and the difference between steady-state holding current before and during external exposure to nine concentrations (range, 0.01-1,000 ±M) of DHO was measured and normalized to cellular membrane capacitance. The concentration dependence of the inhibition of Na-K pump current was biphasic and well fitted to a two-binding site model with inhibitory KDvalues of 0.05 ±M and 64.5 ±M. This is consistent with previously reported3H-ouabain binding studies in guinea pig myocardium. These findings indicate that the electrogenic properties of the Na-K pump can be inhibited by glycoside binding to both high- and low-affinity sites.