Hyperprolactinaemia due to macroprolactin (MPRL) can lead to misdiagnosis and inappropriate treatment. We studied the new Roche Elecsys Prolactin assay (PRL II) which has been developed to reduce reactivity with MPRL. We investigated the performance of the PRL II assay at six laboratory sites to determine precision and establish reference intervals for total immunoreactive PRL and for monomeric PRL determined by precipitation with polyethylene glycol (PEG). We compared the reactivities with macroprolactin of the PRL II, the PRL I and seven other PRL assays. The PRL II assay reacted less strongly than the PRL I assay and similarly to the ADVIA Centaur assay with macroprolactin. PEG precipitation can be used with the PRL II assay to estimate the concentration of monomeric PRL.