Axenic HK9 Entamoeba histolytica strain amoebae, maintained in PEHS medium, displayed several cystic characteristics that involve an active process of cystic wall formation, cellular volume and density diminution, and one or two nuclear divisions. The differentiation process was asynchronic, beginning after the logarithmic growth phase. The axenic cysts, which were maintained in a 50 mOsm/kg medium at 4 degrees C for 72 h, produced growing trophozoites within 1-7 days of incubation at 36 degrees C in fresh medium. Negative results were obtained with trophozoites submitted to the above treatment, and with axenic cysts maintained in double-distilled water at 4 degrees C for 24 h, or in 0.1% sarkosyl, for 10 min at room temperature instead of 55 mosmol/kg medium. Thus, the HK9 E. histolytica strain, cultured in PEHPS, produced under axenic conditions a small proportion of mature, metabolically active cysts, but with an immature or abnormal wall.