Background: The limitations of serum testosterone and estradiol (E 2 ) measurements using nonextraction platform immunoassays (IAs) are widely recognized. Switching to more specific mass spectrometry (MS)-based methods has been advocated, but directly comparative data on the two methods are scarce. Methods: We compared serum testosterone and E 2 measurements in a large sample of middle-aged/elderly men using a common platform IA and a gas chromatography (GC)-MS method, in order to assess their limitations and advantages, and to diagnose male hypogonadism. Of subjects from the European Male Aging Study (nZ3174; age 40-79 years), peripheral serum testosterone and E 2 were analyzed using established commercial platform IAs (Roche Diagnostics E170) and in-house GC-MS methods. Results: Over a broad concentration range, serum testosterone concentration measured by IA and MS showed high correlation (RZ0.93, P!0.001), which was less robust in the hypogonadal range (!11 nmol/l; RZ0.72, P!0.001). The IA/MS correlation was weaker in E 2 measurements (RZ0.32, P!0.001, at E 2 !40.8 pmol/l, and RZ0.74, P!0.001, at E 2 O40.8 pmol/l). Using MS as the comparator method, IA ascertained low testosterone compatible with hypogonadism (!11 nmol/l), with 75% sensitivity and 96.3% specificity. The same parameters with IA for the detection of low E 2 (!40.7 pmol/l) were 13.3 and 99.3%, and for high E 2 (O120 pmol/l) 88.4 and 88.6%. Conclusion: A validated platform IA is sufficient to detect subnormal testosterone concentrations in the diagnosis of male hypogonadism. The IA used for E 2 measurements showed poor correlation with MS and may only be suitable for the detection of high E 2 in men.