OBJECTIVE: We describe a case where interference by heterophile antibodies in a cortisol immunoassay led to a falsely lowered serum cortisol level. CASE DATA: As part of a diagnostic workup for fatigue, a 42-yr-old woman was found to have a low 9.00 h serum cortisol and an abnormal Synacthen stimulation test consistent with adrenal insufficiency. On insulin hypoglycemia testing, markedly discrepant cortisol levels were obtained from paired samples tested by two different immunoassays. Further tests, including plasma ACTH and overnight metyrapone test, confirmed normal hypothalamic-pituitary-adrenal axis function. Negative interference on the initial immunoassay from a heterophile antibody in the patient's serum was detected, thereby explaining the abnormal results. CONCLUSIONS: This report demonstrates that potentially significant consequences can arise from the failure to consider heterophile antibody interference. Clinicians who interpret test results need to be aware of the potential for interference in immunoassays by heterophile antibodies.