Thiamine triphosphate can be found in most tissues at very low levels, but its role is unknown. Organs and muscles that generate electrical impulses are particularly rich in this compound. This paper describes a thiamine triphosphatase from the electrical organ of Electrophorus electricus. The activity of this enzyme, as measured by a high-performance liquid chromatographic method, is closely anion-regulated. Furthermore, thiamine triphosphate increases chloride uptake in membrane vesicles prepared from rat brain. Our results suggest that this compound could play an important role in the regulation of chloride permeability.