A procedure for visualizing and quantifying motility of Entamoeba histolytica by migration under agarose is described. Agarose suspended in tissue culture medium 199 supplemented with bovine albumin was poured into plastic dishes and allowed to harden. Six pairs of wells were cut out in a circular configuration. To the inner wells a suspension of E. histolytica in Eagle's Medium (24 × 106 cells/ml) was added, and to the outer wells a chemoattractant or the control medium. After overnight incubation at 37°C, the amebae were fixed and stained. The chemotactic and spontaneous migrations were measured in an enlarging projector. Escherichia coli filtrates, suspensions of intact and lysed erythrocytes, and the complement factor C5a acted as good chemoattractants. Both the random and chemotactic motility were correlated to the time of the incubation. Cytochalasin B effected a dose-related inhibition of both chemotactic and random migration, while colchicine caused a decrease of the chemotaxis only. The reproducibility of the method, measured by 10 intra-assay tests, was good. Thus, the described method can be useful for comparative determinations of the motility of different ameba populations. Furthermore, different factors affecting the motility of amebae can be studied.