Monoclonal antibodies (MAbs) of two different specificities were produced by immunizing mice with the semipurified M antigen of histoplasmin. One type, from clone CB4, was an immunoglobulin M that precipitated a polysaccharide present in histoplasmin and also formed immunoprecipitates with a cross-reactive polysaccharide present in extracts of Blastomyces dermatitidis and Coccidioides immitis. The second type of MAb, from clone EC2, was an immunoglobulin G that reacted in the enzyme-linked immunoelectrotransfer blot (EITB) assay with a doublet of proteins with an apparent molecular size of 70 to 75 kilodaltons. This molecule is proposed as the authentic M protein antigen that is recognized by M antibodies in sera from mice and rabbits immunized with Histoplasma capsulatum and from persons with histoplasmosis. The M factor also occurs in an abundant disulfide-bridged dimer which has a molecular size of 150 kilodaltons and is nonimmunoreactive under the conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.