1. Formulae developed by Anfinsen et al. (1946) for a medium used in the cultivation of Plasmodium knowlesi were used for media in which excystation of Entamoeba histolytica without bacteria was investigated.
2. The following media were used: (i) an inorganic fluid containing carbon dioxide and chlorides, phosphates, and bicarbonates, of sodium, potassium, magnesium, and calcium; (ii) an inorganic fluid of chlorides and phosphates of the above metals without bicarbonates; (iii) the same respective fluids plus glucose, and (iv) the same plus enrichments with B vitamins, cocarboxylase, vitamin C, purines, pyrimidines, folic acid, glucosamine, cholesterol, and amino-acids. The oxygen content of all media was lowered either by cysteine or glutathione.
3. Some excystation occurred in all media, the percentages were lowest in the inorganic fluid without bicarbonates, highest in fluid with bicarbonates plus all of the listed organic compounds, and intermediate in the inorganic bicarbonate fluid plus glucose. Amino-acids were not required for good percentages of excystation.
4. Excystation occurred when cysts were isolated in medium with Trypanosoma cruzi.
5. The data show that organic compounds in the medium are necessary for high percentages of excystation.