The purpose of this study was to evaluate the variation in plasma adrenocorticotropic hormone (ACTH) concentration and dexamethasone suppression test (DST) results with season, age, and sex in healthy, pony mares (n=15) and pony stallions (n=14) living under semiferal conditions and horse mares (n=10) living at pasture. Plasma ACTH concentrations were measured in September 2002, and in January, May, and September 2003. DSTs were performed in January and September 2003. Plasma ACTH concentrations in September 2002 and September 2003 were similar and were significantly greater than in January and May (P < .001). Plasma ACTH concentration was within the reference range for 38 (97%) of 39 subjects in January, for 39 (100%) of 39 subjects in May, for 2 (5%) of 39 subjects in September 2002, and for 3 (8%) of 39 subjects in September 2003. DST results were within the reference range in all subjects in January and were within the reference range for 29 (74%) of 39 subjects in September 2003. Plasma cortisol concentration at the end of the DST was significantly greater in September than in January (P = .002). Age was positively correlated with plasma ACTH and plasma cortisol concentration at the beginning and end of the DST Within the same season, plasma ACTH concentration in pony mares, pony stallions, and horse mares was not significantly different (P > .05). Seasonal changes in plasma ACTH concentration and DST results should be considered when interpreting endocrine test results.