A simple and rapid method for extracting specific cell-free antigens (CFA) from the yeast form of Paracoccidioides brasiliensis grown on agar slants was developed. Extracts were analysed mainly by immunodiffusion (ID) tests, for the immunodiagnosis of paracoccidioidomycosis. Extracts were obtained from 2 to 7-day-old cultures. The extract obtained after the third day, which contained 200-300 micrograms of protein/ml, gave the best ID reactions, with 100% sensitivity and specificity. The main precipitation band was intense and showed total identity with that formed by the specific 43 kDa glycoprotein. Other tests such as counterimmunoelectrophoresis and immunoelectrophoresis were also used successfully with CFA. The 3-day CFA extract showed a pattern of polypeptides in the 10-110 kDa range after SDS-polyacrylamide gel electrophoresis. Five components of 20, 43, 45, 55 and 70 kDa reacted on immunoblots with sera from patients with active chronic paracoccidioidomycosis. The specific diagnostic antigen of 43 kDa always predominated in all CFA preparations. The present method is thus very useful for the rapid production of an antigenic extract which can be readily characterized and used in the serodiagnosis of paracoccidioidomycosis.