Cells of a newly described, immortalized, epithelial, human transverse colonic cell line, NCM460, reach ~90% confluence on plastic and develop transepithelial resistances of 120-250  · cm² on porous substrates. Its utility as a model for the transverse human colon was validated by comparing second messenger-mediated Cl transport, using the fluorescent probe 6-methoxy-quinolyl acetoethyl ester, in NCM460 cells and colonocytes isolated from human transverse crypts. Basal Cl influx was increased (P < 0.01) by PGE₁ (1 µM), forskolin (1 µM), 8-bromoadenosine 3'5'-cyclic monophosphate (100 µM), heat-stable Escherichia coli enterotoxin (STa; 1 µM), 8-bromoguanosine 3'5'-cyclic monophosphate (100 µM), histamine (1 µM), and phorbol 12,13-dibutyrate (1 µM) in both cell types. The Cl channel blocker diphenylamine 2-carboxylic acid (50 µM) and the Na⁺-K⁺-2Cl cotransport inhibitor furosemide (1 µM), but not the K⁺ channel blocker Ba²⁺ (3 mM), inhibited these Cl permeabilities. These cells possess transcripts for cystic fibrosis transmembrane conductance regulator, Na⁺-K⁺-2Cl cotransporter, STa receptor, and intestine-specific cGMP-dependent protein kinase II. Thus cAMP-, cGMP-, and Ca²⁺-dependent secretagogues act on NCM460 and primary colonocytes to stimulate Cl transport. This validates the utility of NCM460 as a model for transverse colonic crypts and is the first demonstration of a colonic cell line whose origin is known.