Dysfunction of the autonomic nervous system is of prognostic value for sudden death after acute myocardial infarction. Although the use of beta-blockers to counteract the adrenergic hyperactivity has been shown to decrease mortality in these patients, there have been no reports on the role of cholinomimetic drugs in the prognosis of patients after myocardial infarction. The present study was designed to investigate the effect of the administration of pyridostigmine bromide, a reversible anti-cholinesterase agent, on cardiac cholinergic activity assessed by the resting and reflex heart rate responses. Eight healthy volunteers were submitted to a conventional 12-lead electrocardiogram to obtain resting heart rate, and to three non-invasive cardiovascular tests: respiratory sinus arrhythmia, Valsalva maneuver and 4-sec exercise test. On two different days and following a randomized cross-over double-blind protocol, the experiments were performed before and 120 min after oral administration of either pyridostigmine bromide (30 mg) or placebo. Pyridostigmine increase (P < 0.05) the duration of the R-R intervals at rest (pre: 898 +/- 30 msec; post: 1019 +/- 45 msec; pre-placebo: 916 +/- 26 msec; post: 956 +/- 28 msec; P > 0.05). Although the duration of the R-R intervals during the autonomic tests was also increased (P < 0.05), the derived indexes of maximal fluctuation during the maneuvers did not change. These results indicate that oral pyridostigmine produces tonic cardiac cholinergic stimulation while exerting no effect on its reflex changes. Further studies are needed to address the potential role of the administration of pyridostigmine in the prognosis of patients with acute myocardial infarction.