Abstract— Methionine enkephalin (met‐enk), an endogenous opioid, commonly occurs in sequences of lipotropins (LPH) and endorphins, implying a possible biosynthetic pathway for this pentapeptide. In search of the enzyme which generates met‐enk from human β‐endorphin [LPH(61‐91), β‐end], it was found that the soluble fraction of rat brain contained such activity. The enzyme was purified by ammonium sulfate fractionation (50‐80% saturation), ion‐exchange chromatography on DEAE‐Sephadex A‐50, and affinity chromatography for which LPH(64‐67) functioned as affinity ligand and eluant. The final preparation was essentially free of other peptidases, such as kininase, met‐enk degrading and post‐proline cleaving activities. Studies on specificity revealed that the enzyme selectively cleaved the Met‐Thr bond in both LPH(64‐67) and β‐end. It is possible that this enzyme participates in the biosynthesis of met‐enk in vivo.