The biophysical properties of cloned ion channels are often studied by characterising the conductance conferred upon HEK293 cells by heterologous expression of wild type / mutant channel protein. Whilst this approach clearly depends upon the assumption that HEK293 cells do not express any significant background conductance, voltage-gated ion channels have been described in these cells (e.g. Moran et al. 2000; Smirnov et al., 2003; He & Soderland, 2010). Since these endogenous conductances have the potential to complicate studies of cloned channels, the aim of the present study was to characterised the voltage-gated conductances present in this laboratory’s stocks of HEK293 cells in order to facilitate the design of subsequent studies of heterologously expressed channels. Analysis of currents recorded from cells held under voltage clamp (whole cell configuration) using pipette and bath solutions designed to maintain quasi-physiological ionic conditions ([Na+]o = 144 mM; [K+]i = 113 mM) showed that depolarisations (Fig. 1Aa) to potentials >-50 mV evoked outward currents that developed over ~20 ms (Fig 1Ab). Iso-osmotically replacing bath Na+ with K+ had little effect upon these outward currents but did evoke a transient “tail current” that became apparent when Vm was repolarised (ITail, Fig. 1Ac). Experiments in which the K+ content of Na+ free bath solutions was varied by iso-osmotically substituting N-methyl-D-glucammonium (NMDG+) showed that ITail always reversed at a potential essentially identical to EK (Fig 1B). Moreover, replacing pipette K+ with Cs+ consistently (n = 7) abolished the depolarization-induced outward current. Further examination of currents recorded under standard conditions (Fig 1Ab) showed this voltage-induced outward current was preceded by a transient (~10 ms) inward current that persisted after internal K+ had been replaced by Cs+ (n = 7). This inward current became evident once Vm was depolarized past -50 mV, and reversed at a potential essentially identical to ENa. Moreover, its magnitude was greatly (~95%) reduced if bath Na+ was replaced with NMDG+ (Fig. 1C). HEK293 cells thus express voltage-gated K+ and Na+ channels that allow depolarizing voltage pulses to evoke a transient, inward Na+ current that is succeeded by a sustained outward K+ current. Although derived from the human kidney, it is therefore clear that HEK293 cells display a phenotype reminiscent of a neurone rather than an absorptive epithelial cell. Moreover, the presence of these voltage-gated conductances has the potential to interfere with the characterisation of cloned channels expressed in these cells.