Ultrastructural analysis of Entamoeba histolytica reveals that this intestinal human pathogen lacks recognizable mitochondria, but the presence in its genome of genes encoding proteins of mitochondrial origin suggests the existence of a mitochondrially derived compartment. We have cloned the full-length E. histolytica gene encoding one such protein, chaperonin CPN60, and have characterized its structure and expression. Using an affinity-purified antibody raised against recombinant protein, we have localized native E. histolytica CPN60 to a previously undescribed organelle of putative mitochondrial origin, the mitosome. Most cells contain only one mitosome, as determined by immunofluorescence studies. Entamoeba histolytica CPN60 has an amino-terminal extension reminiscent of known mitochondrial and hydrogenosomal targeting signals. Deletion of the first 15 amino acids of CPN60 leads to an accumulation of the truncated protein in the cytoplasm. However, this mutant phenotype can be reversed by replacement of the deleted amino acids with a mitochondrial targeting signal from Trypanosoma cruzi HSP70. The observed functional conservation between mitochondrial import in trypanosomes and mitosome import in Entamoeba is strong evidence that the E. histolytica organelle housing chaperonin CPN60 represents a mitochondrial remnant.