We have prepared a monoclonal antibody specific for a major 94,000 dalton protein from the brush border membrane of rabbit kidney cortex. The monoclonal antibody was used for the immunoaffinity purification of this protein after solubilization of brush border membranes with octylglucoside. The 94,000 dalton protein is a peptidase capable of cleaving the Gly3-Phe4 bond of methionine-enkephalin. Identification of this peptidase as a previously described 94,000 dalton enkephalinase of kidney cortex was confirmed by its sensitivity to EDTA and inhibitors such as thiorphan and phosphoramidon.