Choline (Ch)-free plasma rats were induced successfully by intravenous (IV) injection of choline oxidase (ChO) (16). However, brain acetylcholine (ACh) levels were not affected by ChO treatment, maintaining the same levels as those in controls, although brain Ch levels were significantly decreased. To clarify the reasons for this, in vivo microdialysis was carried out in the treated rats' striata. The ChO treatment induced not only a 70% decrease of extracellular Ch levels but also a 40% decrease of extracellular ACh levels, reflecting the amount of ACh released from cholinergic terminals. In addition, plasma-bound Ch levels and choline acetyltransferase (CAT) and acetylcholinesterase (AChE) activities in the brain were examined in the rats receiving ChO treatment. No significant differences from controls were observed in these levels. The results suggest that: approximately 70% of striatal extracellular Ch is physiologically supplied from circulating plasma-free Ch; the inhibition of ACh release is related to the maintenance of tissue (intraneuronal) ACh levels under the condition of halting of the supply of free Ch from blood to the brain; if there is a compensative supply of free Ch from de novo synthesis, autocannibalism, or plasma-bound Ch, this may be supplied within neuronal cells, because the level of extracellular-free Ch maintained its depressed level even 11-14 h after ChO treatment.