Improving the husbandry in the southern three-banded armadillo (Tolypeutes matacus) through gaining knowledge of its stress physiology is imperative to maintaining a healthy, zoo-housed population. Our objectives were to: 1) validate the use of fecal hormone analysis for monitoring adrenocortical activity using both an adrenocorticotropic hormone (ACTH) challenge and biological events; and 2) characterize longitudinal adrenocortical activity in male and female southern three-banded armadillos. An ACTH injection was given intra-muscularly to one male (4IU/kg; 5.6IU total) and one female (5.5IU/kg; 8IU total) southern three-banded armadillo. Fecal samples were collected 1day pre- and continued 5days post-ACTH to capture the physiological response measured by elevated fecal glucocorticoid metabolites (FGM) to validate these techniques. Additionally, natural and routine events, including pairing individuals for breeding and veterinary procedures/handling, were used to biologically validate these techniques. To characterize adrenocortical activity, fecal samples (∼3025 total; n=275/animal/yr) were collected from 11 (5 males; 6 females) southern three-banded armadillos 5–7 times a week for 1year at Lincoln Park Zoo (Chicago, IL). A cortisol enzyme immunoassay was used for FGM analysis. The ACTH challenge in the male resulted in a twofold increase of FGM (1123.2±36.2ng/g dry feces) above baseline (675.7±10.0ng/g dry feces) at approximately 54–94h post- injection. The female exhibited a twofold increase (1635.4ng/g dry feces) over baseline FGMs (608.5±12.3ng/g dry feces) approximately 30h post-injection. Reproductive behaviors and veterinary procedures resulted in elevated FGM concentrations from all individuals except for one male. The longitudinal characterization demonstrated that sex and season did not influence (P<0.05) FGM concentrations. Individuals were highly variable with mean FGM concentration of 2010.1±862.4ng/g dry feces (range, 816.3–7889.1ng/g dry feces). Mean FGM baseline concentration was 878.5±201.8ng/g dry feces (range, 475.2–1955.5ng/g dry feces) with a mean elevated FGM concentrations of 2694.3±1111.4ng/g dry feces (range, 1110.3–10,683.3ng/g dry feces). This study provides the foundation for future research on how the environment directly affects the adrenocortical activity in this species of armadillo.