Absorption of short-chain fatty acids has been studied in the proximal and the distal colon of anaesthetized guinea pigs. Segments were perfused with a solution similar in chemical composition to that of normal colonic fluids. In the proximal colon the permeability of the mucosa was similar for acetate, propionate and butyrate. For acetate the permeability was significantly higher in the proximal than in the distal colon, and the reverse was seen for butyrate. In the distal colon the short-chain fatty acids seem to be absorbed mainly in the undissociated form due to their lipid solubility; a paracellular pathway for the dissociated molecules is of no major importance. In the proximal colon, on the other hand, a considerable portion of acetate and propionate disappears in the ionized form. Light microscopy (semithin sections) and electron microscopy (freeze-fracture replicas) showed remarkable morphological differences between the proximal and the distal colon. "Leaky spots" with only few strands were present in the zonulae occludentes between the epithelial cells at the surface of the proximal colon. In the distal colon the junctions between the cells were more compact, and significantly more strands separated the lumen from the intercellular space. These results suggest that short-chain fatty acids could be absorbed by a paracellular pathway in the proximal colon, and not in the distal colon. In the proximal colon the number of strands of the zonulae occludentes between surface cells and that between cryptal cells was similar. On the contrary, in the distal colon significantly more strands were present between surface cells than between cryptal cells. Morphological and physiological considerations suggest that absorption of short-chain fatty acids in the crypts is negligible.