Regional cerebral acetylcholine (ACh) levels and utilization rate were assessed in vivo in rats rendered thiamin deficient using the thiamin antagonists pyrithiamin or oxythiamin. ACh levels were significantly reduced in all brain regions of pyrithiamin treated rats and in the medulla-pons and striatum of oxythiamin treated rats compared to controls. ACh utilization was significantly reduced in the midbrain, striatum and hippocampus of pyrithiamin treated rats, but was reduced only in the striatum of oxythiamin treated rats compared to controls. Thus, there are some reductions in ACh levels and utilization that are unique to pyrithiamin induced deficiency and as such are distinct from oxythiamin/undernutrition related reductions. Since only pyrithiamin produces neurological symptoms, its unique ACh effects may be related to these symptoms.