Dog fibroblasts grown from a biopsy performed in a male mongrel were fused after gamma irradiation with thymidine kinase-deficient hamster cells and cultivated in selection medium. A total of 148 clones were obtained and screened by means of PCR amplification using primers corresponding to a dog-specific short repetitive element and to dog microsatellites and genes. One hundred seven cell lines were selected and grown in roller bottles and the distribution of 39 markers was analyzed in the extracted DNA. The results clearly indicate that this panel of hybrid cell lines should prove invaluable for constructing a map of the canine genome. In parallel, for more than 500 microsatellites present in the databases or screened from two libraries of short inserts, we have determined PCR conditions favoring dog-specific products even in the presence of hamster DNA. These highly polymorphic microsatellites should be useful in further linkage studies. We have also characterized 254 markers: dog genes, human expressed sequenced tags (huESTs), and traced orthologous amplified sequenced tags (TOASTs). Once mapped, these will constitute powerful tools to detect regions of conserved synteny in human and other mammalian genomes.