The adrenal is characterized by the production of three groups of hormones, i.e. mineralocorticoids, glucocorticoids and the so called, adrenal androgens. These steroids exhibit independent regulation and are produced within separate adrenal compartments. The zona glomerulosa, fasciculata and reticularis represent three phenotypically different cell types that produce aldosterone, cortisol and DHEA-sulfate as a result of very different steroidogenic enzyme expression profiles. Adrenal diseases that lead to adrenal steroid excess are often due to enzyme deficiencies or neoplastic processes. Both events cause a dramatic alteration in normal adrenal zonation and the expression pattern of steroid metabolizing enzymes. Recent genomic studies of normal and pathologic adrenal tissues have shown that adrenal adenomas and cancer have enzyme expression profiles that would predict the production of novel steroid hormones. Likewise, enzymatic deficiencies would predict the production of unique hybrid steroids not seen in normal physiology. Based on these studies we have expanded our use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to test the hypothesis that neoplastic adrenal tissue and CAH results in circulating steroids that could act as biomarkers for patient diagnosis. While the concept of adrenal disease steroid biomarkers is not a new concept, the increased sensitivity and availability of LC-MS/MS has provided physicians access to panels of steroids that will improve diagnosis of common and rare adrenal diseases. This seminar will discuss the University of Michigan Adrenal Program’s quest to define serum steroid biomarkers for primary aldosteronism, congenital adrenal hyperplasia and adrenal cancer.