The cascade of cellular events that is triggered by low O2 levels in the central nervous system depends on initial sensing mechanisms that can be crucial in determining the overall cell response, adaptation, or injury. In this report, we demonstrate that the activity of an identified K+ channel is regulated directly by environmental O2. Membrane ionic currents were recorded from neurons of the neocortex and the substantia nigra and studied by using whole-cell or excised membrane patches. O2 deprivation reversibly induced an initial transient increase in whole-cell outward currents, and this was followed by a pronounced decrease in these currents. In cell-free excised membrane patches, lack of O2 reversibly inhibited a class of K+ channels that are inhibited by ATP and activated by Ca2+. K+ channel inhibition depended on pO2 level, with a 50% inhibition at approximately 11 torr (1 torr = 6.9 kPa). By the use of specific agents that chelate metal in metal-containing O2-sensing centers, including heme, nonheme iron, copper, and flavin, we also demonstrated that iron-center but not copper-center blockers inhibited the channel in excised patches in a similar fashion as low pO2. These results strongly suggest that K+ channel activity is modulated during O2 deprivation by nonheme iron-containing proteins that are associated with channel molecules, thus providing evidence for a direct O2-sensing mechanism in neuronal membranes.