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 "Ca(2+)-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.