Cysts of Entamoeba histolytica obtained from 2 asymptomatic subjects were cloned in vitro and the isoenzyme patterns and virulence of the cultures derived from them were determined. Incubation in Diamond's TYI-S-33 medium with Crithidia sp. for 80 d resulted in a change from zymodeme III to zymodeme II, with consistent virulence. Axenization by replacing Crithidia sp. with crithidial lysate had no further effect on the zymodeme but the virulence of the amoebae was attenuated. A reversal to zymodeme III, together with augmentation of virulence, was observed on the 20th day after transfer of axenic amoebae to Diamond's TYSGM-9 medium containing the original bacterial flora. The possibility that the isoenzyme pattern determining the zymodeme and the virulence behave as 2 independent variables in E. histolytica is discussed.