CONTEXT:Heterophilic antibodies are a well-described interferent but poorly appreciated and are often not a recognized problem affecting most immunoassays. We describe for the first time heterophilic antibodies interference affecting an adrenocorticotropic hormone (ACTH) assay in a patient with Cushing's syndrome due to bilateral nodular adrenal hyperplasia. CASE:A 60-year-old retired female nurse underwent extensive invasive investigations, which were ultimately unnecessary, as a result of initial analytical interference in the ACTH assay, which could not be resolved using a proprietary heterophilic binding reagent. RESULTS:This case highlights the inherent difficulty of diagnosing Cushing's syndrome and the large emphasis placed on laboratory tests. The consequence of not initially identifying interference in this patient's laboratory test results led to unnecessary and costly investigations with potentially adverse outcomes. CONCLUSIONS:Clinicians and the laboratory community need to be continuously vigilant and view laboratory results with caution when they are inconsistent with the clinical picture. This approach is paramount, especially at a time of increasing automation and ever-diminishing scientist involvement in sample processing.