Rats were treated acutely (one day) and chronically (seven days) with atropine, paraoxon, or both, and brain levels of acetylcholine (ACh) were determined to elucidate possible mechanisms underlying adaptation to chronic cholinesterase (ChE) inhibition. In acutely treated animals, atropine had no effect on free ACh, but decreased bound ACh levels. Conversely, paraoxon increased both free and bound ACh. Atropine pretreatment totally prevented the paraoxon-induced rise in free and bound ACh. In chronically treated animals, atropine decreased total ACh content after the initial injection, with no further changes upon subsequent administration. ACh levels increased during the first three days of paraoxon treatment, but latter injections (days 4-7) did not produce further alterations. Daily pretreatment with atropine prevented the paroxon-induced increase in ACh levels on days 1-3, but could not prevent the paraoxon-induced rise in ACh on days 4-7. Results are discussed in terms of a possible neurotransmitter accumulation, occurring under conditions of chronic ChE inhibition, that may regulate the mobilization, storage, and release of ACh.