Depending on its severity and duration, O2 deprivation activates mechanisms that can lead to profound deleterious changes in neuronal structure and function. Hypoxia also evokes inherent adaptive mechanisms that can possibly delay injury and increase neuronal survival. One of these neuronal adaptive mechanisms is believed to be the activation of K+ channels, but direct evidence for their activation is lacking. We performed experiments to test the hypothesis that hypoxia induces activation of K+ channels via changes in cytosolic and membrane factors such as ATP, Ca2+, and membrane potential. The effect of hypoxia on single-channel currents was studied in rat substantia nigra neurons, since these have a high density of glibenclamide binding sites. In cell-attached patches, hypoxia or cyanide reversibly activated an outward current. This hypoxia-activated current in excised inside-out patches was K+ selective and voltage dependent, and had a high sensitivity to internal ATP, ADP, and AMP-PNP, a nonhydrolyzable ATP analog. Activation of this channel required the presence of free Ca2+ on the cytosolic side, but charybdotoxin or apamin did not have any effect on this channel. The effect of ATP on channel activity was not a result of Ca2+ chelation because Mg.ATP in high Mg2+ background and K2.ATP in high Ca2+ environment inhibited the channel. These results suggest that although this hypoxia-activated K+ channel shares properties with ATP-sensitive K+ (KATP) channels in other tissues, substantia nigra neurons seem to have a different subtype or isoform of KATP channels. Gating this channel by multiple factors simultaneously would allow this channel to be particularly suitable for activation during metabolic stress.