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A complement-fixing (CF) antigen was prepared from amastigotes and trypomastigotes of Trypanosoma cruzi (Ernestina strain) grown in beef embryo cell cultures. Multiple lots of the antigen, which consisted of a supernate of washed and disrupted organisms, required material from 106 to 107 total organisms per ml for optimum CF activity. Antibody at dilutions up to 1:256 was demonstrable in various sera from infected animals or patients. Contaminating beef cells from infected cultures were shown to be partly responsible for cross-reactions of the antigen by CF with sera from cases of cutaneous leishmaniasis in whom concomitant infection with T. cruzi could be excluded. There were no cross-reactions with syphilitic sera and the frequency of positive reactions with normal sera was very low. Some characteristics of the antigen included stability to storage at -20° C and -70° C for months, inactivation at 60° C and by lyophilization, and an estimated molecular size of between 50,000 and 100,000 on the basis of membrane filtration.