The relationship between physostigmine (Phy) concentration, acetylcholine (ACh), choline (Ch) and cholinesterase (ChE) activity was examined in whole rat brain after the administration of [3H]Phy (650 microgram/kg i.m.). Cholinesterase inhibition was found to be inversely related to Phy levels. Maximal inhibition (80%) was seen at 5 min and by 2 hrs ChE activity had returned to control levels. Acetylcholine levels in whole brain peaked at 30 min at a concentration (80 nmol/g) 2.3 times higher than controls (33 nmol/g). Choline levels were not significantly altered. The regional distribution of Phy concentration and ChE activity was studied in six areas of the brain following i.m. administration of three different dosages of ( 3H]Phy. Physostigmine concentration and ChE activity showed a dose dependency in each area examined except in SP (medial septum). Striatum (ST) showed the greatest relative increase of ACh up to 30 min, when compared to other areas. Choline levels were not changed in any area with the exception of ST at 5 min where a decrease was seen. There was a relationship between ChE activity, Phy concentration and ACh levels in all areas examined with exception of the medulla oblongata (MO). Our results indicate that even though ChE was inhibited practically uniformly in all brain areas, the percent increase with respect to control animals and the relative increase of ACh varied widely from area to area. This finding has clinical implications in cases in which cholinomimetic therapy is used to elevate ACh levels in specific brain areas which show a cholinergic deficit.