The basic properties of microsomal thiamine diphosphatase (TDPase) in rat brain were examined. It was mainly localized in the microsomal fraction. Microsomal TDPase showed an absolute requirement for divalent cation which was best satisfied by Ca⁺⁺. Theophylline (1mM), NADP (0.1mM) and NADPH (0.1mM) significantly inhibited its activity. However, caffeine, theobromine, NAD and various putative neurotransmitters did not affect the enzyme activity. UDP was inhibitory and IDP was a competitive inhibitor, but ATP had no effect on the enzyme activity.