Two monoclonal antibodies (MAb I and IV) have been prepared which showed high and specific reactions towards bovine heart mitochondrial coupling factor B (FB). Both have been identified as sub-type IgG1 of mouse immunoglobulins. MAb I reacts with purified and functionally active FB, alkylated or oxidized forms of FB and even with peptides formed on digestion of FB with trypsin. When used together, MAb I and IV reacted with FB in immunoblots of normal and urea treated samples of mitochondria, submitochondrial particles, ammonia-EDTA extracted particles, and H+-ATPase. Both MAbs inhibited FB-stimulated ATP-dependent reverse electron flow activity when FB was incubated with the antibody either before or after its addition to FB-deficient AE-particles. Reactivity of MAb I towards FB declined upon exposure of FB to guanidine HC1 while reactivity of MAb IV remained unaltered.