Whole-cell fusion between zebrafish fibroblast-like ZF4 cells and mouse B78 melanoma cells resulted in hybrids containing one or a few zebrafish chromosome segments in a murine chromosomal background. Fluorescence in situ hybridization to hybrid cell metaphases with a zebrafish genomic DNA probe revealed that many hybrids contained zebrafish chromosome segments that were either inserted or translocated to a mouse chromosome, whereas other hybrids contained zebrafish chromosomes with no evidence of insertion or translocation. We have assigned hybrids to 17 linkage groups of the genetic map of the zebrafish genome. Our results demonstrate the feasibility of producing somatic cell hybrids between distantly related species. Zebrafish/mouse cell hybrids will provide a useful tool for the physical mapping of the zebrafish genome and for the cloning of genes affected in zebrafish mutants.