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Niacin (nicotinic acid) was found to be essential for the cultivation of axenic Entamoeba histolytica. This vitamin requirement was also satisfied by nicotinamide. Panmede liver digest, a source of vitamins in the axenic medium was replaced with a dialyzed hot water extract of homogenized whole liver, supplemented with various growth factors. Culture medium made with the liver extract and not supplemented with niacin failed to support continued multiplication of E. histolytica, but did support serial subculture when niacin was added. The concentration of added niacin required to achieve maximum growth was about 1 µg per ml of medium. This is the first demonstration of a niacin requirement by the organism.