Paneth cells in small intestinal crypts secrete microbicidal -defensins in response to bacteria and bacterial antigens (Ayabe et al, Nature Immunol 1:113, 2000). We now report that the Ca²⁺-activated K+ channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium activated and was blocked by ten structurally diverse peptide and non-peptide inhibitors with potencies spanning seven orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (~50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is an important modulator of Paneth cell -defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.