The purpose of this study was to determine whether nitric oxide (NO) contributes to the hypotensive state induced by prolonged environmental heat (EH) stress. Ketamine-anesthetized rats were instrumented for the measurement of arterial blood pressure, electrocardiogram, and temperature at four sites. Rats were exposed to EH (ambient temperature, 40 ± 1°C) until mean arterial blood pressure (MAP) decreased to 75 mmHg, which was arbitrarily defined as the induction of heatstroke. In addition to cardiovascular and temperature measurements, the time required to reach this MAP end point and the subsequent survival time were measured. In three separate experimental series, the competitive NO synthesis inhibitor N-nitro-L-arginine methyl ester (L-NAME) was administered (0, 10, or 100 mg/kg) either before, during (30 min after initiation of EH), or immediately after EH. L-NAME administered at any of these times transiently increased MAP. L-NAME infusion either before or during EH did not alter the EH time required to decrease MAP to 75 mmHg, but L-NAME pretreatment did decrease the colonic temperature at which this MAP end point was reached. L-NAME infusion before or after EH did not affect subsequent survival time, but L-NAME administered during EH significantly decreased survival time. The administration of L-NAME at any time point, therefore, did not prove beneficial in either preventing or reversing heatstroke. Taken together, these data suggest that NO does not mediate the hypotension associated with heatstroke.