In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven 86Rb+ fluxes through K+ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba2(+)-sensitive, Ca2(+)-activated K+ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5 x 10(-7) M free Ca2+. This suggests an important role of cytoplasmic Ca2+ in the regulation of the bidirectional ion fluxes in the colon epithelium. The properties of K+ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca2(+)-activated K+ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantly in the crypt cell membrane fraction. Titrations with H+ and tetraethylammonium show that both high- and low-sensitive 86Rb+ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba2(+)-sensitive, Ca2(+)-activated K+ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.