A small-conductance K+ channel in the apical membrane of rat cortical collecting duct (CCD) cells controls K+ secretion in the kidney. Previously, we observed that the activity of the channel is stimulated by cAMP-dependent protein kinase A (PKA)-induced channel phosphorylation. We now have applied the patch-clamp technique to study the effects of protein kinase C (PKC) on the secretory K+ channel of rat CCD. In cell-attached patches, application of phorbol 12-myristate 13-acetate progressively reduced the open probability and current amplitude of the K+ channel. In inside-out patches, administration of PKC reversibly decreased the channel open probability (Po) without changing the channel conductance. The PKC-induced inhibition of channel activity was Ca2+ dependent: Po decreased 42%, 23%, and 11% in the presence of 1000 nM, 100 nM, and 10 nM free Ca2+, respectively. We also demonstrate that PKC antagonizes the stimulatory effect of PKA on the apical K+ secretory channel of rat CCD. These results suggest regulation of K(+)-channel activity by two separate sites of phosphorylation with distinct and opposite effects on channel activity.