Abstract The Na/K pump current I p of single HEK293 cells either untransfected (endogenous I p) or transfected with the α1 subunit of the rat Na/K pump (exogenous I p) was investigated in Na-containing solution by means of whole-cell recording at 30°C. The endogenous I p was irreversibly blocked by 10 −4 M ouabain or 2·10 −4 M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF −1 at 0 mV and 5.4 mM K o. It was half maximally activated at 1.5 mM K o and increased linearly with depolarization over the entire voltage range studied (−80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant α1 subunit of the rat Na/K pump showed an I p in the presence of 10 −4 M ouabain and 2·10 −4 M DHO, respectively. This exogenous I p was reversibly blocked by 10 −2 M ouabain. Half maximal activation of the exogenous I p occurred at 1.7 mM K o. Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the I p of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous I p in HEK293 cells is generated by the α1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording.