Endocannabinoids are a family of endogenous signaling molecules that modulate neuronal excitability in the central nervous system (CNS) by interacting with cannabinoid (CB) receptors. In spite of the evidence that astroglial cells also possess CB receptors, there is no information on the role of endocannabinoids in regulating CNS function through the modulation of ion channel-mediated homeostatic mechanisms in astroglial cells. We provide electrophysiological evidence that the two brain endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) markedly depress outward conductance mediated by delayed outward rectifier potassium current (IK(DR)) in primary cultured rat cortical astrocytes. Pharmacological experiments suggest that the effect of AEA does not result from the activation of known CB receptors. Moreover, neither the production of AEA metabolites nor variations in free cytosolic calcium are involved in the negative modulation of IK(DR). We show that the action of AEA is mediated by its interaction with the extracellular leaflet of the plasma membrane. Similar experiments performed in situ in cortical slices indicate that AEA downregulates IK(DR) in complex and passive astroglial cells. Moreover, IK(DR) is also inhibited by AEA in NG2 glia. Collectively, these results support the notion that endocannabinoids may exert their modulation of CNS function via the regulation of homeostatic function of the astroglial syncytium mediated by ion channel activity.