To evaluate the pituitary-adrenal axis function by means of the adrenocorticotropic hormone (ACTH) stimulation test following a single intravitreal injection of triamcinolone acetonide (IVTA). Prospective comparative clinical interventional study. Twenty-eight patients (28 eyes) received a single IVTA (4 mg in 0.1 ml) for macular edema. The basal cortisol level and the response to 1 μg adrenocorticotropic hormone stimulation were determined on the morning before IVTA injection and at 1 day and 1, 2, and 4 weeks after IVTA injection. Results were compared with those obtained from a control group of 50 healthy subjects. All patients in the study had normal basal cortisol and normal response to ACTH challenge before receiving IVTA. 1 day following IVTA, basal cortisol was suppressed in one patient in the study group. Fasting serum cortisol levels at 1, 2, and 4 weeks after IVTA injection were normal in all patients in the study group. 1 day following IVTA, the peak response to ACTH at 30 min was blunted in four patients (14.3 % of the study group, p = 0.05) and the cortisol response at 60 min was suppressed (p = 0.009). 1 week following IVTA, the response to ACTH challenge was blunted in only one patient. A single IVTA injection may be associated with impaired hypothalamic-pituitary-adrenal function in some patients during the first 24 h following IVTA.