Using a yeast two-hybrid screen of a B-cell cDNA library with an Epstein-Barr nuclear antigen 5 (EBNA5) molecule containing seven repeats of the W(1)W(2) domain as bait, we have isolated the EBNA5-interacting protein HAX-1. HAX-1 has previously been shown to associate with HS1, a protein specifically expressed in cells of the haematopoietic lineage, and is thought to be involved in signal transduction in B-cells. Immunofluorescence experiments showed that HAX-1 co-localized with the hsp60 protein that is associated with the mitochondria in the cell cytoplasm. Pull down experiments with a fusion protein between glutathione S-transferase and the seven copy repeat EBNA5 synthesized in bacteria and in yeast cells confirmed that HAX-1 can interact with EBNA5 in vitro. Conventionally, EBNA5 is regarded as a nuclear protein. However, we show here that the smallest EBNA5 species, composed of the unique Y domain and only one copy of the W(1)W(2) repeat domain, like HAX-1, co-localizes with the mitochondrial hsp60 protein in the B-cell cytoplasm. Furthermore, immunoprecipitation experiments demonstrate that the single repeat EBNA5 associates with HAX-1 in transfected B-lymphoblastoid cells.