Chromatograms prepared from interrenal incubates or blood from two species of cartilaginous fish consistently failed to show the presence of cortisol, corticosterone, or aldosterone but did reveal a substance, more polar than cortisol, which gave reactions expected of a corticoid. The substance was identified as the previously unknown 1α,11β,21-trihydroxypregn-4-ene-3,20-dione. Dehydration converted the steroid to the known 11β,21-dihydroxypregna-1,4-diene-3,20-dione. The NMR spectra confirmed the location of the hydroxy group and established the α-(axial) configuration. The steroid crystallized in needles from acetone-hexane, m.p. 201–202°, λ max methanol 242.5 mμ, ε, 15,600. It formed a diacetate, m.p. 182–183°. The sulphuric acid chromogen and IR spectra of the new steroid and its 1-dehydro derivative are presented. 1α,11β,21-Trihydroxypregn-4-ene-3,20-dione was produced in interrenal incubates from endogenous precursors (e.g. 6.4 μg/100 mg/hr with added ACTH) and from [4- 14C] corticosterone. Other products were formed only in trace amounts. From 1150 ml of blood plasma 18 μg of the steroid were isolated. A few characteristics of the 1α-hydroxylating enzyme system are briefly discussed.