Many proteins including ion channels are regulated by phosphorylation. We tested the effect of 10 U/ml catalytic subunit protein kinase A on 260-pS Ca(2+)-activated K+ channels in excised inside-out membrane patches from freshly dispersed smooth muscle cells of the canine proximal colon. At +50 mV with 10(-7) M Ca2+ and -50 mV with 10(-6) M Ca2+, open probability of the channels was increased to 270 +/- 48% of control (n = 12). This increase was due to a shift in voltage-dependent activation by 13.9 +/- 3.2 mV (n = 3) to more negative potentials. Protein kinase A in the absence of ATP had no effect on channel activity (n = 3). Regulation by phosphorylation must be accompanied by dephosphorylation. We tested the effect of two potent inhibitors of protein phosphatases, calyculin A and okadaic acid. Application of 10(-9) to 10(-6) M of each inhibitor in the presence of protein kinase A further increased open probability by up to 250%. Calyculin A appeared to be less effective in increasing open probability than okadaic acid, suggesting that the phosphatase involved is neither type 1, 2A, nor 2B. Calyculin A in the absence of protein kinase A was ineffective. These data suggest that endogenous phosphatases are found in excised membrane patches and that a balance between phosphorylation and dephosphorylation may provide an important control of colonic motility.