A haemolytic agent (haemolysin), which lysed rabbit erythrocytes in an isotonic buffer. occurred in the digestive section of the midgut of adult male and female tsetse flies (Glossina morsitans) but not in the anterior, nondigestive section of the midgut. Haemolysin from females also lysed horse, cow, and pig erythrocytes, and it occurred in the midgut lumen but not in the midgut wall 6, 24. and 48 h after feeding. Little or no haemolysin was present in unfed flies but it was present in the midgut by 3 h after feeding on a rabbit: maximum activity occurred in the midgut 24–48 h after feeding. Flies fed on a meal containing 20 μg or more puromycin/ml produced only small quantities of haemolysin. Flies fed upon erythrocytes, haemoglobin, methaemoglobin, and myoglobin produced large quantities of haemolysin while those fed upon erythrocyte stroma, serum proteins, cytochrome c, or 1 mM ATP in 0.85% NaCl did not.