Cryptdins are antimicrobial peptides secreted by Paneth cells located at the base of intestinal crypts. In addition to their antimicrobial function, cryptdins may also regulate salt and water secretion by intestinal epithelial cells. Recent work with short-circuit current measurements indicated that at least one cryptdin peptide, cryptdin 3, induces apical conductance(s) in Cl(-) secretory, including cystic fibrosis, epithelia. In the present study, we characterized the cryptdin 3-induced anion channel activity in human embryonic kidney (HEK) cells with single-channel patch-clamp techniques. The patch pipette was filled with solution containing different concentrations of cryptdin 3, and, after gigaseal formation, the channel activity was recorded with either cell-attached or inside-out patch modes. We found an anion selective channel with a conductance of 15 pS and open probability of 0.19, regardless of cryptdin 3 concentration. The mean open and closed times varied with the cryptdin 3 concentration. For cryptdin 3 concentrations of 10, 4, 1, and 0.5 microg/ml in the pipette, the corresponding mean open times were 1.2, 7.0, 9.0, and 17.4 ms and the corresponding mean closed times were 1.1, 1.6, 4.2, and 12.5 ms. These results suggest that cryptdin 3 forms anion-selective channels on the cytoplasmic membrane of HEK cells and that the kinetics of one such channel are affected by its interaction with other such channels.