Previous studies have shown that ₂ adrenoceptor (₂AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of ₂AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that ₂AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (I_sc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that ₂AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl^- secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 µg ml^-1), was used to investigate the effect of UK 14,304 on basolateral K⁺ transport. The Na⁺-K⁺-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 µM). In the presence of a basolaterally directed K⁺ gradient, UK 14,304 significantly reduced nystatin-activated I_sc indicating that activation of ₂ARs inhibits basolateral K⁺ channels. Studies with selective K⁺ channel inhibitors and openers showed that ₂AR agonists inhibited K_ATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle K_ATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by ₂AR agonists required activation of pertussis toxin-sensitive G_i/o proteins, but did not involve classical second messengers, such as cAMP or Ca²⁺. In summary, ₂ARs inhibit anion secretion in colonic epithelia by acting on basolateral K_ATP channels, through a process that does not involve classical second messengers.