To obtain purified H and M antigens suitable as primary standards in the serological diagnosis of histoplasmosis by agar gel double-diffusion tests, H and M reactive components of histoplasmin were fractionated by column chromatography by using Sephadex G-100, Sephadex G-200, and diethylaminoethyl cellulose. Six fractions from diethylaminoethyl cellulose were reactive in agar gel double-diffusion, complement fixation, and capillary precipitin tests. When examined by electrophoresis on acrylamide gel, one M antigen (fraction 2, molecular weight greater than 200,000) and two H antigens (fractions 5 and 6, molecular weight greater than 200,000) each gave essentially a single protein band. In agar gel double-diffusion and complement fixation tests with sera from patients with proven cases of histoplasmosis, blastomycosis, or coccidioidomycosis, these two fractions of H antigen and the one of M antigen reacted only with sera from proven or suspect cases of histoplasmosis and showed reactivity with those sera known to contain only the anti-H or anti-M antibody, respectively. Fraction 2 (M antigen) and fractions 5 and 6 (H antigens) had carbohydrate-to-protein ratios of 1.60, 0.77 and 0.78, respectively. Both antigens contained galactose, glucose, mannose, and hexosamine, with mannose being the predominant sugar. Fraction 2 was characterized by a high proline and glucose content, whereas fractions 5 and 6 contained higher concentrations of galactose, mannose, glycine, and alanine. Each of these products appeared to separate into two active fractions, one of a molecular weight greater than 200,000 and one of a molecular weight less than 35,000. The M antigen component of fraction 2 was still characterized by a high proline content, whereas the H antigen components of fractions 5 and 6 had a high content of glutamic acid, serine, glycine, and proline.