Isolated intestinal epithelial cells of the guinea-pig were used to study uptake and metabolism of inorganic sulfate in the jejunum (proximal cells) and ileum (distal cells). Proximal enterocytes accumulated sulfate 1.5-fold and distal enterocytes 3.1-fold. Accumulation was almost linearly related to substrate concentrations up to 5 mM. In proximal cells, despite their lower intracellular sulfate levels, sulfate incorporation into acid-precipitable material and sulfate conjugation of 1-naphthol were faster than in distal cells. Formation of 1-naphthyl sulfate increased with extracellular sulfate concentrations up to 1 and 3 mM sulfate in distal and proximal enterocytes, respectively. The data suggest that the extent of intestinal sulfation of phenolic compounds may be enhanced by oral administration of sulfate.