Endopeptidase EC 18.104.22.168 (EP 24.15; thimet oligopeptidase) is a soluble metalloendopeptidase implicated in the metabolism of a number of neuropeptides, including neurotensin, gonadotropin-releasing hormone, and opioid peptides. We have shown previously that thiol reducing agents, such as dithiothreitol, activate EP 24.15 by mediating the conversion of inactive multimeric forms to active monomers and that this conversion involves the disruption of intermolecular disulfide bonds involving cysteine residues 246, 248, and 253. We have identified two components of cerebrospinal fluid that activate recombinant EP 24.15, but have no effect on a thiol-independent cysteine mutant form of the enzyme. The low molecular weight (50 kDa) is sensitive to digestion with trypsin, suggesting it is proteinaceous in nature. These results suggest that EP 24.15 activity in the brain may be modulated by factors released into cerebrospinal fluid.