The effects of subcutaneous Nociceptin/Orphanin FQ (N/OFQ) administration on corticosterone (CORT) secretion were determined in male and female wild-type mice and mice lacking the N/OFQ prepropeptide. Additionally the effect of pretreating animals with isoflurane anesthesia to minimize the potential stress of injection was examined. Although N/OFQ itself did not specifically increase CORT levels in males or females of either genotype, injection alone (either vehicle or N/OFQ) or isoflurane exposure both increased CORT levels in all groups. These results demonstrate that N/OFQ does not elevate circulating CORT levels; however the injection process itself results in increased CORT secretion. Pretreatment with isoflurane did not significantly diminish the CORT response to injection, except in wild-type males. In fact, isoflurane exposure alone increased CORT levels above basal values. Additionally, a gender difference was evident; females displayed a greater change of plasma CORT levels than males. Finally, because even saline injection increased CORT levels, we closely investigated another possible non-specific stress effect, i.e. the effect of transporting animals from their home environment in the animal facility to the laboratory on the day of the experiment. Although basal CORT levels were similar to values reported in other studies, circulating CORT levels were elevated in animals transported to the laboratory, even after a 30 min acclimation period. These results indicate that the experimental protocol that is followed when conducting stress experiments needs to be carefully considered.