O ₂ deprivation induces membrane depolarization in mammalian central neurons. It is possible that this anoxia-induced depolarization is partly mediated by an inhibition of K⁺ channels. We therefore performed experiments using patch-clamp techniques and dissociated neurons from mice neocortex. Three types of K⁺ channels were observed in both cell-attached and inside-out configurations, but only one of them was sensitive to lack of O₂. This O₂-sensitive K⁺ channel was identified as a large-conductance Ca²⁺-activated K⁺ channel (BK_Ca), as it exhibited a large conductance of 210 pS under symmetrical K⁺ (140 mM) conditions, a strong voltage-dependence of activation, and a marked sensitivity to Ca²⁺. A low-O₂ medium (PO₂ = 10–20 mmHg) markedly inhibited this BK_Ca channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O₂ deprivation on BK_Ca channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pH_i), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BK_Ca channel activity by reducing the sensitivity of this channel to Ca²⁺. In contrast, the reducing agents glutathione and DTT increased single BK_Ca channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BK_Ca is modulated by O₂ deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca²⁺ sensitivity resulting from cytosolic alternations such as in pH_i and phosphorylation. Because of their large conductance and prevalence in the neocortex, BK_Ca channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.