Meprins are oligomeric, glycosylated cell surface or secreted metalloendopeptidases that are composed of multidomain disulfide-linked subunits. To investigate whether subunit oligomerization is critical for intracellular transport or for the enzymatic and/or physical properties of the proteinase, specific cysteine residues were mutated, and the mutants were expressed in 293 cells. Mutation of mouse meprin alpha Cys-320 to Ala in the MAM domain (an extracellular domain found in meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu) resulted in expression of a monomeric form of meprin, as determined by SDS-polyacrylamide gel electrophoresis and nondenaturing gel electrophoresis. The monomeric subunits were considerably more vulnerable to proteolytic degradation and heat inactivation in vitro compared with the oligomeric form of the enzyme. Proteolytic activity of the monomeric meprin using a bradykinin analog or aminobenzoyl-Ala-Ala-Phe-p-nitroanilide as substrate was similar to that of disulfide-linked oligomeric meprin; however, activity against azocasein was markedly decreased. Mutation of another cysteine residue in the MAM domain (C289A), predicted to be involved in intrasubunit disulfide bridging, resulted in disulfide-linked oligomers and monomers. These results indicated that this mutant was capable of forming intersubunit disulfide bonds but less efficiently than wild-type meprin subunits. Mutant C289A also retained activity toward peptides but not the protein substrate and was more vulnerable to proteolytic degradation and heat inactivation compared with the wild-type enzyme. Both Cys mutants were expressed and secreted into the medium at levels comparable with the wild type and had slightly altered glycosylation. This work indicates that 1) Cys-320 of mouse meprin alpha is most likely responsible for the covalent interactions of the subunits; 2) covalent dimerization of subunits is not essential for efficient biosynthesis, trafficking, or posttranslational processing of the secreted protease; and 3) mutations in the MAM domain affect noncovalent interactions of the subunits and the stability and activity of the protease domain, indicating that domain-domain interactions are critical for structure and function of the enzyme.