Host blood effects on Trypanosoma congolense establishment in Glossina morsitans morsitans and Glossina morsitans centralis were investigated using goat, rabbit, cow and rhinoceros blood. Meals containing goat erythrocytes facilitated infection in G.m.morsitans, whereas meals containing goat plasma facilitated infection in G.m.centralis. Goat blood effects were not observed in the presence of complementary rabbit blood components. N-acetyl-glucosamine (a midgut lectin inhibitor) increased infection rates in some, but not all, blood manipulations. Cholesterol increased infection rates in G.m.centralis only. Both compounds together added to cow blood produced superinfection in G.m.centralis, but not in G.m.morsitans. Midgut protease levels did not differ 6 days post-infection in flies maintaining infections versus flies clearing infections. Protease levels were weakly correlated with patterns of infection, but only in G. m. morsitans. These results suggest that physiological mechanisms responsible for variation in infection rates are only superficially similar in these closely-related tsetse.