A synthetic, channel-forming peptide, derived from the α-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH₂ terminus (N-K₄-M2GlyR). In Ussing chamber experiments, apical N-K₄-M2GlyR (250 μM) increased transepithelial short-circuit current (I _sc) by 7.7 ± 1.7 and 10.6 ± 0.9 μA/cm² in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672–C1679, 1997). N-K₄-M2GlyR caused a concentration-dependent increase inI _sc (k _[1/2] = 190 μM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K₄-M2GlyR-mediated increases in I _sc were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl⁻ conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4′-dinitrostilben-2,2′-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K₄-M2GlyR-mediatedI _sc. Whole cell membrane patch voltage-clamp studies revealed an N-K₄-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K₄-M2GlyR forms a 50-pS anion conductance with a k _[1/2] for Cl⁻of 290 meq. These results indicate that N-K₄-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.