The regional distribution of thiamine and its phosphate esters was measured in the central nervous system (CNS) of normal and thiamine-deficient mice. Twelve small areas were punched out from frozen sections and they were individually analyzed by high performance liquid chromatography (HPLC). Regional difference was noted in both the content and ratio of thiamine and its phosphate esters in the normal CNS. In pyrithiamine-induced thiamine deficiency, thiamine pyrophosphate (TPP) content in all the areas was reduced to less than 13% of the control values on day 10, when the neurological signs developed. Although there were considerable regional variations in the reduction rate of thiamine and its phosphate esters, no correlation was established between the severity of tissue damage and the magnitude of thiamine reduction in individual areas. These results indicate that a derangement in TPP-dependent processes, either alone or in combination with other factors, plays a more critical role in the neuronal damage under thiamine deficiency than depletion of thiamine compounds per se.