High conductance apical K⁺ (BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well-fitted to contribute to the excessive intestinal K⁺ losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K⁺ enrichment. We found that PKA activation using 50 µmol/L forskolin and 5 mmol/L 3-isobutyl-1-methylxanthine (IBMX) stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The anti-diarrheal peptide somatostatin (SOM; 2 µmol/L) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pre-treatment with 10 µmol/L okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A), but completely prevented by pre-treatment with 100 µmol/L Na⁺ orthovanadate and 10 µmol/L BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K⁺ losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new anti-diarrheal strategies.