BACKGROUND In 2005, a special survey in the Belgian External Quality Assessment focused on the performance of six automated immunoassay analysers most frequently used in Belgium for estradiol-17β (E 2 ) and progesterone. Results obtained were compared with values determined by reference method, isotope dilution–gas chromatograph/mass spectrometry (ID–GC/MS). METHODS Five fresh frozen serum samples, without additives, from single donors and three pools from pregnant women were distributed to all registered Belgian laboratories. Total variation, bias, linear relationship within the reported range and linear regression were investigated. RESULTS Inter-laboratory coefficients of variation ranged from 4 to 49% for E 2 and from 6 to 45% for progesterone. Bias ranged from −26 to 239% for E 2 and from −23 to 81% for progesterone. Several systems showed an upward bias for one particular sample of at least 25%. Weighted linear regression showed overall bias ranging from −8% to 32% for E 2 and from 7% to 41% for progesterone. CONCLUSIONS Few automated methods succeed in having an excellent reproducibility for E 2 and progesterone. Given the high bias values it is suggested that, for performance testing, results be compared whenever possible with a reference method. The linear relationship as assessed by comparing results with those obtained by ID–GC/MS using samples from different donors was not assured for most methods.