Five men underwent eight heat stress tests (HSTs) at 35 degrees C, each consisting of four 25-min treadmill walks (35% Vo2max), separated by 5-min rests, in four conditions: 1) 20% relative humidity (rh), subjects euhydrated and drinking ad libitum; 2) 20% rh, euhydrated; 3) 75% rh, euhydrated; and 4) 20% rh, hypohydrated 3% of body weight. In Conditions 2-4 subjects drank during the walks to maintain their pre-HST weight. In each condition we tested subjects once after 30 mg pyridostigmine bromide (PB) by mouth and once after placebo. PB lowered heart rate a mean of 3 beats/min overall, most with hypohydration. PB did not significantly affect rectal temperature (Tre), but reduced the rise in Tre during hypohydrated exercise. In Condition 2, chest skin temperature decreased more during exercise with PB. PB had no significant effect on other skin temperatures, sweating, hematocrit, hemoglobin, total plasma protein, osmolality, ad libitum drinking, rate of O2 uptake, or subject ratings of temperature, discomfort, or exertion. PB thus had little effect on physiological responses to moderate exercise-heat stress.