The autonomic imbalance in experimental models of hypertension is characterized by an increase in sympathetic and decrease in parasympathetic function. While sympathetic overactivity has received most of the attention, the same appraisal has not been given to the decrease in parasympathetic function. Therefore, it was investigated whether an increase in the availability of acetylcholine will improve the sympathovagal balance during the development of hypertension in SHR. To accomplish this, it was performed the blockade of central acetylcholinesterase with donepezil (DON), a compound that crosses the blood-brain barrier, and also pyridostigmine (PYR), a compound which does not cross the blood-brain barrier, acting particularly in the peripheral synaptic cleft. Thus, it was investigated the chronic effect (16 weeks) of DON or PYR on the following variables: arterial pressure (AP); heart rate (HR); vagal tone; AP and pulse interval (PI) variability in time and frequency domain; plasma norepinephrine (NE) and cardiac function. Acetylcholinesterase blockade started when SHR were 5 weeks old, receiving DON (1.5 mg/kg/day) or PYR (2mg/kg/day) given by osmotic minipump. After 16 weeks, femoral AP was recorded in conscious state and atropine provided the vagal tone, while the combined administration of atropine plus propranolol provided the intrinsic HR (iHR). Cardiac function was assessed under isofluorane anesthesia, with the Millar catheter, 24h after the hemodynamic measurements. Systolic AP (Control: 202±5; DON: 178±8; PYR: 200±7 mmHg) and HR (Control: 354±13; DON: 304±9; PYR: 334±10 bpm) was reduced by DON, but was not affected by PYR. Vagal tone was increased by DON or PYR (Control: 64±5; DON: 105±11; PYR: 132±8 bpm). PYR increased the iHR while DON did not (Control: 340±3; DON: 351±5; PYR: 381±8 bpm). DON decreased the systolic AP variance (Control: 63±5; DON: 39±7) and the power of the low-frequency (LF) band of PI (Control: 32±2; DON: 18±2 nu), while decreased the power of the high-frequency (HF) band (Control: 68±2; DON: 82±2 nu) and the LF/HF ratio (Control: 0.5±0.05; DON: 0.2±0.03) of the PI spectrum. PYR did not promote significant changes in the AP or PI variability. Moreover, DON or PYR did not affect NE concentration (Control: 8.7±0.7; DON: 12±2.2; PYR: 9.6±2 ng/mL) or cardiac function [+dPdt (Control: 9469±574; DON:11461±1156; PYR:9640±1041 mmHg/s), -dPdt (Control: -11665±1085; DON: -11015±570; PYR: -9999±1294 mmHg/s), end diastolic pressure (Control:9±2; DON: 11±3; PYR: 7±1 mmHg)]. In conclusion, despite the increase in cardiac vagal tone elicited by either DON or PYR, the anticholinesterase agent that crosses the blood brain barrier, i.e. DON, was more effective improving the cardiovascular function during the development of hypertension in SHR.