Abstract Rats were given intraperitoneal injections of physostigmine (PHYSO), neostigmine (NEO) or saline (SAL). Either 15 or 30 min later the number and intensity of observable cholinomimetic effects (OCE) was determined, plasma was collected for corticosterone (Cst) measurement, and the cerebral cortex, striatum, hippocampus and hypothalamus were removed after microwave treatment for the measurement of tissue acetylcholine (ACh) and choline (Ch) concentrations. Plasma Cst correlated with the number of OCEs at both 15 and 30 min in both NEO- and PHYSO-treated animals. Although the number and intensity of OCE were the same in NEO- and PHYSO-treated animals 15 min after injection, plasma Cst was significantly higher in the PHYSO-treated group. ACh levels in the cortex were also increased in PHYSO- compared with NEO-treated animals 15 min after injection. Ch levels remained unchanged. Plasma Cst correlated positively with ACh levels in the cortex and striatum in PHYSO-treated rats both 15 and 30 min after injection. These data support the involvement of central cholinergic mechanisms in the regulation of the HPA axis.