Lectin (PHA-P) activated nonadherent spleen cells from uninfected inbred strains of mice known to exhibit high parasitemias when infected with Trypanosoma cruzi (A/J and C3H/HeJ), released in vitro significantly less lymphotoxin (LT) than did a mouse strain (C57BL/6) known to exhibit low parasitemia when infected with T. cruzi. The capacity of mice to release LT in vitro changed upon infection with T. cruzi. Cells from infected C57BL/6 mice released LT levels well above that from uninfected C57BL/6 animals within 4 days after initial infection, and their capacity to release LT remained high even after the parasites were not detectable in the blood. Cells from infected C3H/HeJ mice were not able to respond as rapidly as those fom C57BL/6 animals; however, they were able to release LT at the same levels as the infected C57BL/6 by day 18. The parasitemia induced by Trypanosoma cruzi in C3H/HeJ and C57BL/6 mice was compared with the in vitro ability of their spleen cells to spontaneously release LT. Spontaneous release of LT was significantly higher by spleen cells from infected C57BL/6 mice than by cells from infected C3H/HeJ mice. The kinetics of natural LT release differed from that of mitogen-(PHA-P)-stimulated LT release; LT activity peaked at 3-6 hours of incubation in the former and then declined whereas LT activity in the latter reached a plateau after 3 hours of incubation and did not decline for at least 18 hours in the presence of the inducer. Supernatants from infected C57BL/6 splenocytes, when concentrated 5x by ultrafiltration, significantly inhibited both blood stream trypomastigote motility in vitro and their infectivity (by 98%) for WI38 human embryonic lung cell cultures. Similar preparations from C3H/HeJ splenocytes only slightly inhibited bloodstream trypomastigote motility and delayed, but did not prevent, infection of WI38 cells. No agglutination of immobilized parasites was noted. Splenocyte supernatants did not affect culture epimastigote motility or growth. This is the first report of direct action by lymphokine-containing supernatants against T. cruzi bloodstream trypomastigotes.