The ability to mass cultivate Entamoeba histolytica under axenic conditions is crucial for many types of study on this organism to be undertaken. A major component of the current axenic medium, TYI-S-33, is Casein Digest Peptone. Recently a critical situation arose as no new lots of CDP could be found that supported adequate amebal growth. We have now developed a casein-free medium, YI-S, that supports growth of Entamoeba histolytica and other Entamoeba to the same levels as TYI-S-33. TYI-S-33 was also widely used for cultivation of Trichomonas vaginalis and Giardia. YI-S was tested for its ability to sustain the growth of one isolate of Trichomonas and two of Giardia, again with good results. The availability of a casein-free medium will remove the single greatest source of variability in the axenic culture of E. histolytica. We have continued to examine the taxonomy of the genus Entamoeba at the molecular level. A number of additional isolates and species, including E. polecki an organism with zoonotic potential, have been studied. Significant intraspecific genetic variation has been observed in certain species. In contrast to current dogma, we have found that Entamoeba histolytica appears to have organelles related to the mitochondrion. Sequence and genomic structure analyses of two Entamoeba genes encoding proteins thought to be associated with this organelle are underway.