Spironolactone (Sp) is an aldosterone-antagonist diuretic, that is traditionally used in the treatment of hirsutism due to its antiandrogenic effects. Sp can inhibit ovarian and adrenal biosynthesis of androgens, compete for the androgen receptor in the hair follicle, and directly inhibit 5alpha-reductase activity. The steroid suppressive effects are so variable that the receptor-blocking action is considered the most important mechanism. It is probable for this reason that cortisol, DHA-S levels are not significantly changed with spironolactone treatment, even though androstenedione (A4) levels are decreased. We sought explanations for increased plasma A4 (above 20 nmol/L; reference less than 10.5 nmol/) in patients (n = 5) with Polycystic Ovary Syndrome (PCOS) after treatment with spironolactone (up to 150mg daily). A4 increased from 10.4 nmol/L (range 6.5-16.1) to 47.3 nmol/ L (range 23 - 98), LH from 18.4mU/L (range 4.5 - 42.4) to 16.1 mU/L (range 4.4 - 33.2) and testosterone from 3.8 nmol/L (range 1.5-7.9) to 5.6 nmol/L (range 2.8 - 8.5). The increase in A4 was out of proportion to the other changes, suggestive of assay interference. Indeed, when spironolactone was withdrawn for one month plasma A4 fell below 10.5 nmol/L. A4 was measured by a coated tube immunoasay (Diagnostic Products Corporation, Los Angeles CA 90045-5597). Spironolactone cross-reaction in this assay was reported to be 0.109%. This was based on apparent concentration of A4 of 3.8 nmol/L when 1,000 ng/ml of spironolactone was included in the assay. However, blood concentrations of spironolactone may exceed 5,000 ng/ml. Cross- reactivity data for spironolactone was unreliable but difficult to assess because the steroid is poorly soluble in water. Sp metabolites may also cross react. Sp ia already known to interfere in assays for digoxin and aldosterone. Based on the results here requests for A4 should be accepted in patients with Sp when cross reaction has been excluded.