Electron microprobe analysis of quick-frozen distal colonic epithelium from guinea pig was used to locate the cells responding to secretory stimuli. Concentrations of Na, K, and Cl were similar for cells of surface and crypt in the unstimulated state, 8, 149, and 46 mmol/kg wet weight, respectively. Stimulation of either K and Cl secretion with prostaglandin E2 or K secretion alone with epinephrine increased Na to ~12 mmol/kg wet weight in crypt cells but not in surface cells or cells in the crypt neck. This result supports the location of ion secretory cells in the lower two-thirds of the crypt. In the vacuoles of crypt columnar cells, stimulation of KCl secretion decreased K, S, Mg, and Ca and increased Na and Cl, indicative of the concomitant release of vacuole contents. Mucin granules in crypt goblet cells contained more S and Mg than granules in surface goblet cells. These findings support the concept of differentiation in ion and macromolecular secretory function along the axis from crypt to surface epithelium.