A frequently debated question for studies involving the measurement of stress hormones in rodents is the optimal method for collecting blood with minimal stress to the animal. Some investigators prefer the implantation of indwelling catheters to allow for frequent sampling. Others argue that the implantation of a catheter creates a chronic stress to the animal that confounds stress hormone measures, and therefore rely on tail vein sampling. Moreover, some investigators measure hormones in trunk blood samples obtained after anesthesia, a practice that may itself raise hormone levels. To address these controversies, we 1) compared plasma ACTH and corticosterone (CORT) concentrations in pre- and post-stress rat blood samples obtained via previously implanted vena cava catheters, tail vein nicks, or clipping the tip off the tail, and 2) compared plasma ACTH and CORT in rat blood samples obtained by decapitation with and without anesthesia. Rats sampled via indwelling catheters displayed lower pre-stress ACTH levels than those sampled by tail vein nick if the time to acquire samples was not limited; however, elevated basal ACTH was not observed in samples obtained by tail clip or tail nick when the samples were obtained within 3 minutes. Baseline CORT levels were similar in all groups. After restraint stress, the profile of the plasma ACTH and CORT responses were not affected by sampling method. Decapitation with prior administration of CO₂ or pentobarbital increased plasma ACTH levels ~ 13 and 2 fold respectively, when compared to decapitation without anesthesia. These data indicate that tail vein nicking, tail clipping or indwelling venous catheters can be used for obtaining plasma for ACTH and CORT during acute stress studies without confounding the measurements. However, the elevation in basal ACTH seen in the tail vein nick group at baseline suggests that sampling needs to be completed rapidly (<3 minutes) to avoid the initiation of the pituitary stress response. CO ₂ euthanasia and pentobarbital injection before trunk blood collection cause significant stress to animals, as reflected in the elevated plasma ACTH levels. These results support the use of either chronic vascular cannulae or sampling from a tail vein. However, collection of blood under pentobarbital or CO₂ anesthesia is likely to confound the results of stress studies when ACTH is an important endpoint.