The cultivation of Endamoeba histolytica with Trypanosoma cruzi (Phillips, 1950; Phillips and Rees, 1950) represents the first time this amoeba has been grown successfully in the complete absence of bacteria or bacterial products. As a vehicle for testing amoebicidal agents, the culture has proved to be practicable and reliable (Bradner and Rawson, 1951; Nakamura and Anderson, 1951). Since the trypanosomes die off naturally under the environmental conditions imposed by the Phillips culture, it seems apparent that this culture would be especially suited to experiments envolving attempts to grow the amoeba in association with different organisms. The literature reveals several methods used in the past to test for the amoebiontic (amoeba-supporting) ability of different bacteria. Dobell and Laidlaw (1926) used acid or acriflavin-washed cysts of E. histolytica which were placed in tubes of various media and bacteria thus establishing the amoebae with a new flora. Cleveland and Sanders (1930) used the sterile liver abscesses of amoeba infected cats as a trophozoite source in a similar experiment with single species of bacteria. Chinn, Jacobs, Reardon and Rees (1942) made the most comprehensive search for amoebionts to date. Using bacteria-free cysts for amoeba inoculum twenty-six species of bacteria (41 strains) were tested with regard to their ability to cause excystation and growth of E. histolytica in vitro. A heterogenous group of 14 species of bacteria were found capable of doing so. Jacobs (1950) made a compilation of bacterial amoebionts from several sources. Also, he added four more to the list as a result of his experiments using amoebae growing with Streptococcus hemzolyticus in which the latter was eliminated with penicillin and replaced by other types. Although many organisms have been found capable of sustaining the amoeba in vitro, little information concerning the physiological properties of such organisms has been published. It is generally agreed that since it would be difficult to study the physiology of E. histolytica when other organisms are present, a study of the amoebionts might possibly reveal similarities amongst them which would explain their function in supporting growth of the protozoan. It also seems apparent from the literature that an improved, standardized method of isolating the amoeba for use in floral tests would be helpful. Thus the research described in this paper has three purposes: first, to evaluate the Phillips' method as a source of bacteria-free amoebae in cultural studies; second, to study the major physiological activities of bacterial amoebionts isolated in this laboratory; and third, to indicate similarities in the metabolism of supporting organisms which might be of aid in the search for growth factors for the amoeba.