Procedures for the cultivation of Entamoeba histolytica in CO2-bicarbonate buffer system media are described. A simple medium composed of Hanks BSS, 1.4 percent sodium bicarbonate solution, horse serum and rice powder has proven reliable for the isolation and continued maintenance of strains accompanied by a mixed bacterial flora. Strains have been maintained for periods up to 4 years. Growth and encystment follow a pattern apparently corresponding to that in the intestinal environment. The prompt and vigorous growth response of strains indicates that the system meets the physiological requirements and may even afford a considerable degree of simulation of the in vivo environment. A direct microscopic method of studying cultures has facilitated study of strains under cultivation. Preliminary observations show that basic characteristics are not altered by prolonged cultivation. Strain differences in regard to growth, encystment, migration, response to pH and temperature have been noted. Application to the differentiation of strains is suggested.