Thiamine pyrophosphatase (TPPase) activity was demonstrated by means of cytochemistry and electron microscopy in association with myelinated fibers in the central and peripheral nervous system of the rat. The areas studied included corpus callosum, hippocampus, cerebral cortex, cervical spinal cord and sciatic nerves. In the myelin sheaths, the enzymatic activity was found in 3 locations: (1) within oligodendroglial and Schwann cytoplasmic clefts between myelin lamellae; (2) in the major dense line of myelin; and (3) within the periaxonal space. In addition to this myelin-associated TPPase, enzymatic activity was also observed in specific cytoplasmic localizations in myelinogenic cells. Oligodendrocytes displayed TPPase activity within the nuclear envelope and the endoplasmic reticulum cisternae, whereas Schwann cells displayed TPPase activity within the endoplasmic reticulum and Golgi saccules. The results are discussed in relation to the role that TPPase might play in myelinated fibers, including roles in the conduction of nerve impulses or roles in the maintenance of structural configuration of myelin sheaths.