A number of studies have evaluated the possibility that stress-induced changes in blood-brain barrier permeability enhanced the central effects of the carbamate acetylcholinesterase inhibitor, pyridostigmine. We previously found relatively little evidence of stress-induced changes in the acute toxicity of pyridostigmine in rats using a variety of restraint, forced running and forced swimming stress conditions. In this study, we evaluated the effects of sequential pre-exposure to multiple stressors on the acute toxicity of pyridostigmine. Rats (n = 8 per treatment group) were either un-stressed or stressed by restraint (60 min), forced running (60 min, 15 m/min, 6 degrees incline) and forced swimming (15 min), and then given either vehicle (saline, 1 ml/kg, po) or pyridostigmine (30 mg/kg, po) immediately after the final stressor. Functional signs of cholinergic toxicity (involuntary movements, autonomic dysfunction) were recorded at 0.5, 1 and 2 h after dosing. Body temperature was measured both before stress and 2 h after dosing. Rats were sacrificed immediately after 2-h functional observations to collect tissues (whole blood, diaphragm, frontal cortex, hippocampus and cerebellum) for measurement of cholinesterase activity. Stressed rats treated with pyridostigmine exhibited higher lethality (2/8) compared to unstressed rats given pyridostigmine (0/8). Pyridostigmine elicited classical signs of cholinergic toxicity, but the rats that died did not show increased cholinergic signs and no significant differences in cholinergic signs were noted between treatment groups. Cholinesterase activity was significantly inhibited in blood (47-50%) and diaphragm (80%) following pyridostigmine exposure regardless of stress conditions. Slight but significant inhibition (11-15%) of cerebellar cholinesterase activity was observed following pyridostigmine exposure, but inhibition was not influenced by stress. We conclude that while acute lethality from pyridostigmine may be increased by combined, multiple stressors, increased lethality does not appear due to enhanced cholinergic toxicity or via increased cholinesterase inhibition in either central or peripheral tissues.