Macroprolactin or big-big PRL is, usually, a complex of little prolactin (PRL) with anti-PRL autoantibody. There are some patients with hyperprolactinemia showing a high proportion of serum macroprolactin. However, its clinical significance is not clear. Immunoassays used to determine PRL differ in their ability to detect macroprolactin. Moreover, in recent years, PRL assays have changed from isotopic methods (radioimmunoassays and immunoradiometric assays) to non-isotopic automated immunoassays. The effect of macroprolactin on currently used methods is often unknown. The aim of this work was to study the different reactivity of macroprolactin in two immunoassays systems, Elecsys 2010 and ACS Centaur, and to assess the clinical repercussion of this condition.We studied retrospectively 956 consecutive routine patients. Samples with a PRL value >636 mIU/l (211 samples) were subjected to the polyethylene glycol (PEG) precipitation test to detect macroprolactin, and 2 of them also to gel filtration chromatography for further confirmation. PRL was measured by Elecsys 2010 and, alternatively, by ACS Centaur.By Elecsys 2010, macroprolactin was detected in 19 patients (9%). After removing macroprolactin, PRL levels were within the normal range in every case but one. When original sera from patients with macroprolactin were processed with ACS Centaur. PRL levels were normal or only marginally elevated. The correlation of PRL values in samples with and without macroprolactin assayed by both systems was 0.64 and 0.98, respectively.Nearly 9% of hyperprolactinemic patients detected by Elecsys 2010 may have macroprolactin, but the detection rate obtained using ACS Centaur is much lower. As macroprolactin seems to have minimal clinical relevance, it would be important that the users of PRL assays be aware to what extent macroprolactin interferes with their assays, and have available a validated method, such as the PEG precipitation test, to confirm the presence of macroprolactin.