2-Hydroxyphenazine was isolated from culture supernatant liquid of Pseudomonas aureofaciens by solvent extraction, ion-exchange chromatography, and crystallization.The compound was identified by comparison with chemically synthesized 2-hydroxyphenazine by paper chromatography and electrophoresis, infrared and ultraviolet spectrophotometry, and X-ray diffraction. Some preliminary studies on the biosynthesis of the compound are presented. Enzymatic hydrolysis of epi-progoitrin, the major thioglucoside from crambe seed, at pH 3.0 produced the previously characterized (R)-goitrin and an unknown nitrile. The nitrile was isolated and shown to be (S)-l-cyano-2-hydroxy-3-butene. Measurements were obtained for the yield of (R)-goitrin and (S)-l-cyano-2-hydroxy-3-butene as a function of the pH during the hydrolysis of epi-progoitrin. Hydrolysis of progoitrin from rutabaga seed formed the enantiomeric (S)-goitrin and (R)-l-cyano-2-hydroxy-3-butene. In the Cahn-Ingold-Prelog system epi-progoitrin has the (S) configuration at the asymmetric carbon atom of the aglycon and may, therefore, also be designated as a salt of 2-(S)-hydroxy-3-butenylglucosinolate.