Macrophages and certain batches of sera were essential for extracellular multiplication of in medium 199 with 20% (v/v) calf serum at 37 °C. In mixtures of ‘good’ and ‘bad’ batches of sera, multiplication increased as the proportion of the former was increased. Mixtures of ‘bad’ and ‘intermediate’ sera permitted virtually no growth. Phagocytosis of parasites by macrophages was unaffected by different batches of sera, though reduced in medium 199 alone. Replacement of supernatant medium by medium containing ‘bad’ serum reduced the macrophage infection rate no more than did transfer to medium containing ‘good’ serum. Daily addition of medium conditioned by prior contact with macrophages to cultures of trypanosomes without macrophages resulted in growth at least as good as that in the presence of macrophages. Extracellular replication in medium 199 at 37 °C apparently required at least two factors: ‘M factor’ provided by macrophages, but not required at 28 °C; and ‘S+ factor’ present in some batches of calf serum, essential also at 28 °C but not required by intracellular parasites. Some sera appeared to contain an inhibitory ‘S-factor’.


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