To examine the influence of an established infection on subsequent challenge with another unrelated trypanosome serodeme, cattle were subjected to two challenges, 5 to 6 wk apart, with unrelated isolates of Trypanosoma congolense. The primary infection inhibited the establishment of the second infection despite the initial absence of detectable antibody to the trypanosomes used for the second challenge. This was true whether the second challenge consisted of bloodstream forms of the parasite or metacyclics from infected Glossina m. morsitans. Rechallenge with bloodstream forms resulted in a slight antibody response, which was only detectable by immunofluorescence and was much less than in the challenge controls. Although animals subjected to the second challenge by tsetse flies showed no appreciable increase in parasitemia and, in most instances, no chancre reaction at the site where the tsetse bit, they developed readily detectable neutralizing antibody to the metacyclic trypanosomes. That this interference effect was not the result of specific immunity and required an active infection was confirmed by the finding that when infected animals were treated with Berenil prior to rechallenge, they were fully susceptible to the infection.