Fish traps are a common research and management tool in which fish are subjected to procedures that elicit a stress response in other contexts. The effects of trapping on the stress response of sexually mature, wild rainbow trout Oncorhynchus mykiss were investigated during their upstream spawning migration by measuring concentrations of plasma cortisol, lactate, and glucose. Males had significantly lower basal plasma cortisol concentrations (6.1 ± 0.8 ng/mL [mean ± SE]) than females (21.4 ± 5.9 ng/mL). Similarly, the plasma cortisol response in males was significantly lower than that in females for all experiments. Fish working the barrier before entering the trap had increased concentrations of plasma cortisol. Confinement in the trap also induced a stress response. Plasma cortisol concentrations increased to 185.1 ± 40.9 ng/mL in males and 549.1 ± 60.1 ng/mL in females after confinement for 1 h. After processing, the magnitude of the stress response and the relative duration of recovery was less in fish that were confined longer in the trap. However, resting cortisol concentrations in females were not reached after 40 h of recovery in either group. Recovery to resting concentrations of plasma lactate occurred within 15 h after processing. In contrast, concentrations of plasma glucose remained significantly elevated at 40 h after processing. Postspawning fish had significantly lower plasma concentrations of cortisol, glucose, and lactate following application of an extreme stressor compared with prespawning fish. Based on the results of this study, we conclude that the trapping procedure induces a severe and prolonged stress response in wild rainbow trout.