Complex effects of altering intracellular [Ca²⁺] on M-type K⁺ currents have previously been reported using whole-cell current recording. To study the direct effect of Ca²⁺ on M-channel activity, we have applied Ca²⁺ to the inside face of membrane patches excised from rat superior cervical sympathetic ganglion cells. Ca²⁺ rapidly and reversibly inhibited M-channel activity in 28/44 patches by up to 87%, with a mean IC₅₀ of 100 nM. This effect persisted in the absence of ATP, implying that it was not due to phosphorylation/dephosphorylation. A similar effect was observed in 13/13 cell-attached patches when cells were transiently "Ca²⁺-loaded" by adding 2 mM Ca²⁺ to a 25 mM K⁺ solution bathing the extrapatch cell membrane. These observations provide new evidence that Ca²⁺ can directly inhibit M channels, so supporting the view that Ca²⁺ might mediate M current inhibition following muscarinic receptor activation.