After the onset of an acute episode of arrested circulation to the brain and consequent cerebral hypoxia, EEG changes and modifications of consciousness ensue within seconds. This in part reflects the rapid effect of hypoxia on the neocortex, where oxygen deprivation leads to impaired neuronal excitability and abnormal synaptic transmission. To identify the cellular mechanisms responsible for the earliest changes in neocortical function and to determine their time course, we have used patch-in-slice recording techniques to investigate the effects of acute hypoxia on the synaptic and intrinsic properties of layer 5 neurons. Coronal slices of mouse somatosensory cortex were maintained at 37°C and challenged with episodes of hypoxia (3–4 min of exposure to 95% N₂, 5% CO₂). In recordings with cell-attached patch electrodes, activation of ATP-sensitive potassium channels first became detectable 211 ± 11 sec (range, 185–240 sec; n = 6 patches) after the onset of hypoxia. Similar recording techniques revealed no alterations in the properties of Na⁺ currents in the first 4 min after the onset of hypoxia. The earliest hypoxia-induced disturbance was a marked increase in the frequency of spontaneous EPSCs and IPSCs, which began within 15–30 sec of the removal of oxygen. This rapid synaptic effect was not sensitive to TTX and was present in Ca²⁺-free perfusate, indicating that the hypoxia had a direct influence on the vesicular release mechanisms. The incoherent, massive increase in miniature PSCs would be expected to deplete the readily releasable pool of vesicles in cortical terminals, and to thereby markedly distort the neuronal interactions that underlie normal circuit function.