This study describes the identification of aqueous-soluble antigens in Leishmania promastigotes immunologically and biochemically closely related to the major surface antigen. Proteins from surface-iodinated L. braziliensis braziliensis and L. donovani chagasi promastigotes, extracted and separated by partitioning in the detergent Triton X-114, were analyzed. Immunoblotting of the extracted proteins, using homologous antisera, showed recognition of a 72-kDa labeled, amphiphilic antigen of L. b. braziliensis and a 65-kDa surface antigen of L. d. chagasi. The respective homologous sera also recognized non-labeled hydrophilic antigens, similar in their apparent molecular weights to the major surface antigens. The amphiphilic and hydrophilic antigens of each species were found to share common antigenic determinants, inasmuch as monospecific antibodies that recognized the amphiphilic protein reacted with the hydrophilic antigen. Structural homology was also obtained in the peptide-digestion profiles of the amphiphilic and the respective hydrophilic major antigens. Zymogram assay showed that both amphibilic and hydrophilic fractions displayed proteolytic activity that could be directly attributed to the major L. b. braziliensis and L. d. chagasi antigens. The hydrophilic antigens found in this study are probably not hydrolytic products of the surface antigens and occur in large quantities in the promastigote cytosol.