The metabolism inhibition reaction is a test commonly used for the speciation of mycoplasmas. Cattle infected with Mycoplasma bovoculi produce serum antibodies that are active in metabolism inhibition. For characterization of this reaction, affinity matrixes were prepared from lysates of M. bovoculi bound to a monoclonal antibody that recognizes a 94,000 kDa membrane protein (p94) of the mycoplasma. These matrixes containing purified p94 were used to specifically remove metabolism-inhibiting activity from serum samples of cattle infected with M. bovoculi. The results provide evidence that p94 is the major and perhaps only target for the action of complement-independent, metabolism-inhibiting antibodies. This antibody reactivity may inhibit a vital membrane function associated with the p94 protein.