We investigated the regulation of ATP-sensitive K⁺ (K_ATP) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10⁵ M) activated inward currents in the presence of high external K⁺ (90 mM) at a holding potential of 80 mV in dialyzed cells. Glibenclamide (10⁵ M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10⁷ M) inhibited pinacidil-activated current. 4--Phorbol ester (5 × 10⁷ M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of K_ATP channels was increased by pinacidil, and PDBu suppressed openings of K_ATP channels. When cells were pretreated with chelerythrine (10⁶ M) or calphostin C (10⁷ M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10⁶ M). Acetylcholine (ACh; 10⁵ M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10⁷ M) decreased the effect of ACh. Cells dialyzed with protein kinase C -isoform (PKC) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on K_ATP were blocked in these cells. Immunofluorescence and Western blots showed expression of PKC in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates K_ATP in colonic muscle cells and that the effects of ACh on K_ATP are largely mediated by PKC. PKC appears to be the major isozyme that regulates K_ATP in murine colonic myocytes.