Pluripotent embryonic stem cells (ESCs) possess promising potential for cell-based therapies, but their electrophysiological properties have not been characterized. Here we describe the presence of ionic currents in mouse (m) and human (h) ESCs and their physiological function. In mESCs, tetraethylammonium (TEA)-sensitive depolarization-activated delayed rectifier K+ currents (IK(DR)) (8.6 +/- 0.9 pA/pF at +40 mV; IC50 = 1.2 +/- 0.3 mM), which contained components sensitive to 4-aminopyridine (4-AP) (IC50 = 0.5 +/- 0.1 mM) and 100 nM Ca2+-activated K+ current (IK(Ca)) blocker iberiotoxin (IBTX),were detected in 52.3% of undifferentiated cells.IK(DR) was similarly present in hESCs (approximately 100%) but with an approximately sixfold higher current density (47.5 +/- 7.9 pA/pF at +40 mV). When assayed by bromodeoxyurindine incorporation, application of TEA, 4-AP, or IBTX significantly reduced the proliferation of mESCs and hESCs in a dose-dependent manner (p < .05). A hyperpolarization-activated inward current (I(h)) (-2.2 +/- 0.4 pA/pF at -120 mV) was detected in 23% of mESCs but not hESCs. Neither Na(v) nor Ca(v) currents were detected in mESCs and hESCs. Microarray and reverse transcription-polymerase chain reaction analyses identified several candidate genes for the ionic currents discovered. Collectively, our results indicate that pluripotent ESCs functionally express several specialized ion channels and further highlight similarities and differences between the two species. Practical considerations for the therapeutic use of ESCs are discussed.