Although considerable progress has been made during recent years toward the goal of axenic cultivation of Entazmoeba histolytica (criteria of Dougherty, 1953), the methods thus far developed have provided little information on the growth factors or the metabolism of the amoeba. Rees (1955) has summarized these accomplishments which include cultivation with a number of single species of bacteria, including one that shows little evidence of proliferation during the incubation period of the amoeba (Shaffer and Frye, 1948), and without bacteria in association with Trypanosomna cruzi by Phillips (1950). Shaffer and Sienkiewicz (1952) and Shaffer, Sienkiewicz and Washington (1953) cultivated the amoeba in mince of embryos of the chick without any associated microbial organism. A further advance was made by Nakamura (1952) who obtained some growth of the amoeba in a presumably cell-free medium containing succinoxidase prepared after the method of Stotz and Hastings (1937). However, except in one series which showed contamination with bacteria, the amoebas failed to grow in a number of transfers sufficient to rule out effects of the original inoculum of E. histolytica-T. cruzi. As intimated by Cailleau (1937), in order to demonstrate the suitability of a medium for a protozoan organism its growth should continue undiminished through a minimum of 10 serial transfers. During the past 3 years the method of Shaffer et al with chick-mince medium has been in use, with modifications, in our laboratory because this method holds promise of attaining axenic cultures. In agreement with Shaffer et al (1953), Rees, Reardon, and Bartgis (1954) were unable to grow the amoeba in dialysates of chick-embryo medium, but noted that ingredients of the nondialyzed medium will withstand heating up to 50?C without complete destruction of factors supporting amoebae. However, at higher temperatures, even only slightly above 50?C, the factors supporting amoebas were completely destroyed. The present report was prepared to furnish details of an investigation on which an abstract has already been published (Baernstein, Rees and Bartgis, 1956). To obviate complications inherent in serial transfer of macrocultures (test tube cultures), the technique of microculture was used (Rees, 1942; Rees, Reardon and Bartgis, 1950; Phillips and Rees, 1950). A single washed trophozoite of E. histolytica was transferred by microisolation into medium dispensed into a microtube made from a piece of glass tubing approximately 2 x 55 mm. Although the effects of carry over may be eliminated by this method it has thus far not been adapted