The Δ5-3-ketosteroid isomerase of Pseudomonas testosteroni promotes the conversion of Δ5-3-ketosteroids to the α,β-conjugated 3-ketosteroids by an unusual intramolecular rearrangement involving a cis, cis diaxial proton transfer from the 4β- to the 6β-position. The enzyme is available as a pure protein in two crystalline forms of known unit cell dimensions and crystallographic symmetry (monoclinic and hexagonal). The amino acid sequence of the protomer has been established. Many lines of evidence suggest a mechanism of isomerization involving an enolic intermediate and the participation of an acidic and a basic group of the enzyme in the catalytic process. Single histidyl and aspartyl residues may be involved in the isomerization process and a mechanism for their participation is proposed. Certain β,γ-acetylenic 5,10-seco-3-ketosteroids are converted by the isomerase to the corresponding α,β-unsaturated allene(s) which irreversibly inactivate the enzyme. A partial purification of the soluble isomerase of rat liver has been achieved. These preparations are profoundly and specifically activated by reduced glutathione. The precise mechanism of this effect remains to be established.