Serum thyroglobulin (Tg) measurement is a major means of detecting thyroid cancer recurrence. Unlike anti-Tg autoantibody interferences, heterophile antibody (HAB) immunoassay interferences are not well recognized by laboratorians or clinicians as a Tg assay problem. When HAB interferences occur, they usually result in false positive test results. With the current trend to treat some thyroid cancer patients with radioiodine on the basis of an elevated serum Tg result alone, this has the potential to result in unwarranted therapy. We evaluated the prevalence of HAB interference in a commonly used automated immunoassay in 1106 consecutive specimens with Tg values greater than 1 ng/ml. All Tg measurements were repeated after sample incubation in heterophile-blocking tubes (HBT). Results, which showed a more than 3 SD percentage difference from the original result, were considered to suffer from HAB interference. All possible interferences were confirmed by dilution testing. After HBT treatment, Tg levels dropped to less than 1 ng/ml in 32 specimens (P < 0.0000001), 20 of which fell to less than 0.1 ng/ml (P < 0.00002). Of these 20, 17 were anti-Tg autoantibody negative, and all 32 showed a fall of greater than 3 SD percentage (>56.91%) compared with the original result. There were also two samples that showed a significant increase of greater than 56.91% after HBT treatment. HAB interference is relatively prevalent (1.5-3%) in a commonly used automated Tg assay and can lead to clinically significant artifacts. It is currently unknown, but possible, that other immunometric Tg assays suffer from similar problems. Unless a Tg assay is confirmed to be free of HAB interference or uses additional blocking steps, as ours now does, HAB interference should be suspected if Tg results do not fit the clinical picture.