An avirulent strain (M129-B175) of Mycoplasma pneumoniae is morphologically indistinguishable from its virulent parent strain (M129-B7). Functionally, the avirulent strain has lost its ability to attach to respiratory epithelium and does not produce pneumonia in hamsters. Biochemical analyses by one- or two-dimensional SDS-gel electrophoresis have so far failed to produce evidence that could account for the changes in the avirulent strain. It is possible that proteins of the avirulent strain have been altered by spontaneous mutations to nonfunctional states, events which would not necessarily alter their physical properties, i.e., molecular weight. To examine this possibility, Western blots prepared from proteins of the avirulent strain, separated on SDS-gels, were exposed to a collection of monoclonal antibodies to the virulent strain. Immunoradioautographs showed that two protein bands of the avirulent strain lost their reactivity to the monoclonal antibodies, although overall protein profiles were identical. This preliminary observation suggests that spontaneous mutations that lead to structural changes in protein molecules occurred during the derivation of the avirulent strain.