Hybridomas producing monoclonal antibodies to Rickettsia rickettsii were prepared from mice to investigate the function of rickettsial antigens. Of the 31 reactive hybridoma lines thus far tested for immunoglobulin subclasses, 11 belonged to the IgG2A subclass, 9 to the IgG2B subclass, and 7 to the IgG3 subclass; four did not react with any of the isotyping sera. Five of the antibodies recognized epitopes present on molecules that were presumed to be polysaccharide and heterogeneous in molecular weight. Twenty monoclonal antibodies reacted with a 170,000-dalton antigen, and six precipitated both the 133,000- and 32,000-dalton polypeptides. Only those antibodies to the 170,000- and 133,000-dalton antigens protected mice from challenge with R. rickettsii. Antibodies to these same antigens were detected in sera from patients convalescing from Rocky Mountain spotted fever. All monoclonal antibodies reacted with antigens apparently located on the rickettsial surface. The protective activity of these antibodies was not correlated with their reactivity in complement fixation, enzyme-linked immunosorbent assay, and immunofluorescence tests.