We have developed a panel of whole-genome radiation hybrids by fusing irradiated diploid human fibroblasts with recipient hamster cells. This panel of 168 cell lines has been typed with microsatellite markers of known genetic location. Of 711 AFM genetic markers 404 were selected to construct a robust framework map that spans all the autosomes and the X chromosome. To demonstrate the utility of the panel, 374 expressed sequence tags (ESTs) previously assigned to chromosomes 1, 2, 14 and 16 were localized on this map. All of these ESTs could be positioned by pairwise linkage to one of the framework markers with a LOD score of greater than 8. The whole genome radiation hybrid panel described here has been used as the starting material for the Genebridge4 panel that is being made widely available for genome mapping projects.