The majority of patients with Cushings syndrome are ACTH-dependent, but the diagnosis of ACTH-dependence, and then the differential diagnosis between Cushings disease and the ectopic ACTH syndrome, is dependent on the measurement of ACTH. In the mammalian adult anterior pituitary, POMC is cleaved into a variety of products including the normal ACTH(1-39). In the pars intermedia (present in the human fetus and most mammals), ACTH is cleaved further by the prohormone converting enzyme, PC2, to ACTH 1-17 and ACTH 18-39 (CLIP): ACTH 1-17 (which still has significant ACTH receptor activity) is then processed at its C-terminal to ACTH 1-13 amide (α-MSH). This type of processing of ACTH can occur in ectopic tumours and some pituitary adenomas. These fragments generally do not give signals in ACTH two-site immunoassays, but if they are secreted many-fold in excess to ACTH they will bind to and swamp the individual antibodies without forming the two-site liaison that is necessary for the detection of any intact ACTH present. In addition, patients with aggressive ectopic ACTH secreting tumours, especially small cell lung tumours, often secrete high levels of the ACTH precursors. While it is possible to assess the concentrations of ACTH precursors using a specific two-site ACTH precursor assay, most ACTH assays only detect about 2% of ACTH precursors which may confuse the diagnosis. Thus, plasma from patients with ectopic ACTH-producing tumours may often give erroneous signals in two-site immunoassays for ACTH. Such patients may even be thought to harbour adrenal tumours rather than being ACTH-dependent, and there may be discordance between the levels of ACTH and cortisol. Additionally, the very region of ACTH which is important for adrenal receptor activity, and the target of the initial trypsin-like proteolysis by PC2 in the pars intermedia, is also susceptible to cleavage by other trypsin-like enzymes. This means that the collection and storage of plasma for subsequent measurement of intact ACTH by two-site immunoassay can be a problem. Thus, other more stable and immunogenic co-secreted parts of POMC such as pro-γ-MSH (which can cause gross adrenal hypertrophy and hyperplasia), the joining peptide or lipotrophin, or ACTH precursors might thus be more reliable markers for the diagnosis and differential diagnosis of Cushings syndrome.