Monaghan et al. present convincing data that serum cortisol results derived from a commonly used immunoassay must be interpreted with great caution in metyrapone-treated patients, given the findings of their extensive method comparison study using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These data support conclusions from previous publications which invoke cross-reactivity with cortisol precursors generated by the metyrapone blockade of the cortisol biosynthetic pathway. 11-Deoxycortisol (11DOC) is a likely candidate, and Monaghan et al. show a modest correlation between 11DOC and the difference between the immunoassay and the LC-MS/MS (Pearson’s correlation coefficient of 0.47). The authors state that variation in the data is so great that the assay bias cannot be corrected for by a knowledge of either the metyrapone dose or the serum 11DOC concentration. Based on previous data from a series of metyrapone-treated patients, we support this finding and further concluded that interference from 11DOC alone could not account for the difference between the serum cortisol immunoassay and LC-MS/MS results. We also showed that this is a widespread problem as three commonly used cortisol immunoassays are affected. Initially, we calculated the degree of 11DOC cross-reactivity for each of the cortisol assays by performing spiking studies with 11DOC. We then demonstrated in two metyraponetreated patients that the difference between the immunoassay and the LC-MS/MS serum cortisol results could not be entirely accounted for by the 11DOC concentration given the established cross-reactivity. Data presented in this abstract and unpublished data from additional local metyrapone-treated patients support the hypothesis of Monaghan et al. that this effect is most likely to be seen in adrenocorticotropic hormone (ACTH)-dependent Cushing’s syndrome, as the degree of accumulation of steroid precursors is likely to be a function of both the magnitude of the ACTH drive and the extent of metyrapone blockade. In this way it is not dissimilar to congenital adrenal hyperplasia, where interference in serum cortisol immunoassays is also well described. If the data are available, it would be enlightening to see a correlation between the plasma ACTH concentration and the difference between the serum cortisol immunoassay and LC-MS/MS result in the cohort described in this paper, as well as cross-reactivity data for exogenous 11DOC. While the nature of the interference caused by metyrapone blockade in serum cortisol immunoassays remains elusive, we wholeheartedly support the conclusion of Monaghan et al. that current immunoassay methods for serum cortisol lack the specificity for use in metyrapone-treated patients, and this interference is likely to be more marked in patients with a high ACTH drive.