The effect of artificial gastric fluid (AGF), containing 0.5% pepsin and 0.6% hydrochloric acid, pH 1.8, in distilled water, on the excystation and metacystic development of Entamoeba invadens was examined. Excystation, which was assessed by counting the number of metacystic amoebae after inducing excystation, was enhanced by pretreatment of cysts with AGF for 30 to 60 min at 37 degrees C but not 26 degrees C. Longer exposure of cysts to AGF significantly reduced their viability. Significant enhancement of excystation was observed by pretreatment of cysts with distilled water only at 37 degrees C. In addition, 0.6% hydrochloric acid had a comparable enhancing effect on excystation to AGF. Metacystic development, when determined by the number of nuclei in amoeba, was slightly enhanced by pretreatment with AGF. An artificial intestinal fluid (AIF), containing 1% pancreatin, 1% sodium bicarbonate, and 5% ox bile, pH 8.0, in distilled water, had a significant toxic effect on cysts, where 1% pancreatin had neither an enhancing effect on excystation nor a toxic effect on cysts, whereas 5% ox bile had a toxic effect on cysts. Pretreatment of cysts with AGF followed by AIF had a similar toxic effect on cysts to that by AIF only. These results suggest that gastric fluid but not intestinal fluid at 37 degrees C contributes to enhancing excystation for Entamoeba infection.