We report the chromosomal mapping of 43 loci for 40 randomly isolated mouse liver cDNA clones by linkage analysis in an interspecific backcross of ((C57BL/6J x Mus spretus) x C57BL/6J). The clones were sequenced from both sides and a subset was examined for expression in various mouse tissues. Fifteen of the 40 mapped cDNA clones are either identical or strongly related to known sequences in GenBank, while 25 represent new genes. Additional loci mapped in this cross include 53 simple sequence repeat polymorphisms and 40 restriction fragment length variants from previously characterized cDNA markers. Nine homologous human genes were identified for 7 mouse liver cDNA clones. One clone that maps to mouse chromosome 3 (D3Ucla1) identified a novel homologous segment (synteny) on human chromosome 18q23 (D18S372E). These studies provide linkage mapping and initial characterization of random cDNA clones that may provide a resource for the positional candidate cloning of disease genes.