The aim of this work was to standardize metabolic and cytoplasmic Madurella mycetomatis antigen to be applied in the immunodiagnosis of mycetoma. Growth curves were established growing the mold in two broth media with weekly measures of protein and carbohydrate levels and of dry weight. Sera from immunized rabbits and patient's sera were used in immunoprecipitation to test the antigens. Three weeks old shaken cultures in Sabouraud broth at 37 degrees C were established as the optimal conditions to prepare cytoplasmic and metabolic antigens. Nevertheless, exoantigens were produced since the first week. Metabolic and cytoplasmic antigens presented cross reaction and by immunoblotting they shared 33, 56 and 125 kDa molecular weight proteins. Preparation and use of the metabolic antigens is recommended for the immunodiagnosis of mycetoma when using the counterimmunoelectrophoresis technique.