A simple and highly sensitive assay for the detection of Trypanosoma cruzi antigens by spotting samples on nitrocellulose membrane filters is described. The immunobilized antigens are analyzed by subsequent binding of specific anti-T. cruzi immunoglobulins and secondary enzyme-labeled antibodies. Trypomastigote exoantigens were determined by this technique in the supernatant from cell cultures two days after infection. The T. cruzi circulating antigens present in serum specimens collected from mice infected with the parasite were demonstrable by day 3 of infection, when the parasitemia was still low. Antigenemia was also demonstrable by the dot-immunobinding assay in 14 of 16 congenitally infected patients. No cross-reactivity was observed using sera from healthy individuals, as well as from patients with other related parasitoses. The assay proved to be easy to perform and requires only small volumes of samples.