1. Additional evidence has been presented that the formation of heme in vitro from iron and protoporphyrin is enzyme dependent.
2. An enzyme solution was prepared from the active particulate matter of a chicken erythrocyte hemolysate. The enzyme is soluble in 0.15 M KCl and optimally active at pH 7.9.
3. The rate of heme synthesis was constant over the first 30 minutes and approached maximal values at 3 to 4 hours. At optimal concentrations of protoporphyrin and iron the rate of heme synthesis over the first 30 minutes was proportional to enzyme concentration.
4. The enzyme solution was 89 per cent inactivated after heating at 56 C. for 30 minutes. Optimal stability for 3 hours was at pH 7.4. It was unstable when lyophilized or on storage at 5 C. or -30 C.
5. The enzyme was inhibited by 1 x 10-2 M p-chloromercuriphenylsulfonate and 1 x 10-2 M iodoacetamide.
6. There was a 70 per cent loss of activity after dialysis of the enzyme solution for 22 hours against water. The dialyzed enzyme solution was reactivated with either reduced glutathione or cysteine.
7. The enzyme solution augmented heme synthesis in vitro from δ-amino-levulinic acid, porphobilinogen and protoporphyrin, but not from glycine.