A genomic DNA library from Trypanosoma cruzi, the agent of Chagas' disease, was constructed in the gt11 lambda vector and was screened with serum from a Chagasic patient. Out of 53 positive clones, 23 plaques were purified to homogeneity and 10 different groups were defined by cross-hybridization experiments and by reaction of antibodies selected with products from each recombinant clone. Native T. cruzi proteins of molecular mass ranging from 85 to larger than 205 kDa that share antigenic determinants with products of the recombinant clones were observed in Western blots of parasite extracts. Some of the native proteins were detected in the trypomastigote stage of the parasite, while others were present in epimastigotes as well. The latter result was confirmed for some recombinant clones by hybridization of the cloned DNA with Northern blots of parasite RNA. Clones from each group reacted differently with nine sera from rabbits infected with several T. cruzi strains as well as with eight sera from human patients. Clone 7 was detected by all rabbit sera but not by three human sera. Conversely, clones 1, 2 and 30 were detected by all human sera but failed to be detected by most rabbit sera. We conclude that several proteins from T. cruzi are antigenically active during infection and that some of them differ in their ability to generate antibodies in rabbit or human infections.