Entamoeba histolytica is the protozoan parasite that causes amoebic dysentery and amoebic liver abscesses in humans. For many years it was believed to be a primitive organism because it lacks many typical eukaryotic features including mitochondria. Recently, two genes that in other organisms encode proteins normally found in the mitochondrion have been isolated, giving evidence for the secondary loss of mitochondrial function in E. histolytica. These are the pyridine nucleotide transhydrogenase (PNT) and the mitochondrial chaperonin cpn60 genes. In this study we isolated and characterised a gene encoding a mitochondrial-type heat shock protein 70 from E. histolytica. cDNA and genomic library clones have been isolated and sequenced. Comparison with previously published sequences confirmed the assumption that E. histolytica comes from mitochondrion - bearing ancestors. Southern blot hybridisation revealed there are two copies of the gene in the genome. Northern blot analysis revealed two transcripts hybridising to the mt-hsp70 probe that differ in length and which are induced by heat shock. In addition, an apparently noncoding, polyadenylated RNA that is also induced by heat shock is encoded immediately upstream of the mitochondrial-type hsp70 gene. Expression analysis was also performed in four other Entamoeba species. Partial cpn60, PNT, and mt-hsp70 genes were isolated and the size of the mRNAs and their heat shock induction levels were investigated by hybridisation to these probes. The similarity of the mt-hsp70 amino terminus to those of hydrogenosomal proteins in conjunction with the phylogenetic analyses suggests it is also likely to be targeted to the mitochondrion-derived organelle of E. histolytica known as the mitosome.