The epidemiology of human and animal trypanosomiasis is determined to a large extent by the number of infected tsetse flies in a specific area. In the field, a substantial proportion of infected flies carry mixed trypanosome infections. The way in which these tsetse flies acquire a mixed infection is not fully understood. In particular, the susceptibility of tsetse flies to sequential infection with trypanosomes is not well understood. Accordingly, laboratory studies were made of the effects of age and prior infection on the probability of Glossina morsitans morsitans (Westwood) developing an infection of Trypanosoma congolense and Trypanosoma brucei brucei after feeding on infected mice. Results of these experiments clearly showed that 20–30-d-old G. m. morsitans can still pick up and develop a mature infection in the mouthparts/hypopharynx for T. congolense or in the salivary glands for T. b. brucei. However, their ability to acquire infection was significantly lower compared with teneral flies. Furthermore, 20–30-d-old flies that already carry a mature T. congolense or T. b. brucei infection remained at least as susceptible to a secondary trypanosome infection compared with noninfected flies of the same age. The immunological and epidemiological repercussions of those findings are discussed.