Stress responses play a critical role in the ecology and demography of wild animals, and the analysis of fecal hormone metabolites is a powerful noninvasive method to assess the role of stress. We characterized the metabolites of injected radiolabeled cortisol in the urine and feces of Columbian ground squirrels and validated an enzyme immunoassay for measuring fecal cortisol metabolites (FCM) with a 5 alpha-3beta,11 beta-diol structure by stimulation and suppression of adrenocortical activity and by evaluation of the circadian pattern of FCM excretion. In addition, we also evaluated the impact of capture, handling, and acclimation to the laboratory on FCM. Cortisol is highly metabolized, with virtually none being excreted, and of the radiolabeled cortisol injected, 31% was recovered in urine and 6.5% in feces. The lag time between cortisol injection and its appearance in urine and feces was 4.5 +/- 0.82 (SE) h and 7.0 +/- 0.53 (SE) h, respectively. FCM levels varied over the day, reflecting circadian variation in endogenous cortisol. Dexamethasone decreased FCM levels by 33%, and ACTH increased them by 255%. Trapping and housing initially increased FCM levels and decreased body mass, but these reversed within 3-7 d, indicating acclimation. Finally, FCM levels were modestly repeatable over time (r=0.57) in wild, live trapped, nonbreeding animals, indicating that FCMs provide a measure of the squirrel's stress-axis state. This assay provides a robust noninvasive assessment of the stress response of the Columbian ground squirrel and will facilitate an integration of its life history and physiology.