An Hsp70 gene, HspA3, has been given provisional assignment to chromosome 21 based on the heat induced expression of a 70 kDa protein in a Chinese hamster ovary-human hybrid cell line. This assignment has not been supported by hybridization data or by cloning of the gene. The aim of the current study is to clarify this localization. We have analysed a series of Chinese hamster ovary-human hybrid lines containing human chromosome 21 by two-dimensional gel electrophoresis and Western blot analysis with anti-Hsp70 antibodies. Only one of these, hybrid 72532-X6, was found to contain an additional Hsp70 protein. Three other hybrid cell lines spanning different parts of chromosome 21 did not contain any additional Hsp70 proteins. The Hsp70 protein in 72532-X6 is constitutively expressed and is not heat inducible. Southern blot analysis indicates that this protein is not the major human cognate protein, Hsp73 (Hsp70-8), but may be one of the other cognate Hsp70s. Our data indicate that hybrid cell lines containing human chromosome 21 do not express a human Hsp70, as has been reported previously. The recent report that the hybrid cell line used to make this claim also contains other human chromosome fragments leads us to conclude that an Hsp70 gene is not localized on human chromosome.