We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K_i = 64 μM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P₂) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-γ-S, whereas α, β-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P₂Y₂ purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N^G-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P₂Y₂ receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.