Sixty-nine hybridomas were generated to produce monoclonal antibodies to species of methanogens representing three of the four families accepted at the present time: Methanobacteriaceae, Methanococcaceae, and Methanomicrobiaceae. The antibody of each of 29 hybridomas cross-reacted with a methanogen of the same species or genus as the immunizing (homologous) strain, whereas the antibody of the other 40 cell lines reacted only with the homologous strain. Inhibition-blocking experiments with compounds of known composition and structure were used to define the fine specificity of antibodies of four hybridomas representing the four genera of the methanogens used for immunization. The combining site of the monoclonal antibody against M. thermoautotrophicum delta H examined is specific for a structure in the pseudomurein involving N-acetyl-glucosamine but not the (1-3) linkage or the C-terminus gamma-Glu-Ala of the peptide, both of which are characteristic of pseudomurein, the cell-wall peptidoglycan distinctive of the Methanobacteriaceae. In contrast, the monoclonal antibody to the other methanogen of this family examined, M. arboriphilus DH1, recognizes a determinant involving gamma-Glu-Ala. Thus pseudomurein expresses at least two dissimilar antigenic determinants in different portions of the molecule. The monoclonal antibodies against M. vannielii SB and M. hungatei JF1, whose families do not possess pseudomurein, did not display specificity for analogues of pseudomurein.