A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH) (using an equine RH(5000) panel) and/or by FISH mapping. A comparison of localizations of 713 genes mapped on the horse genome and on the human genome revealed 59 homologous segments and 131 conserved segments. Two of these homologies (ECA27/HSA8 and ECA12p/HSA11p) had not been previously identified. An enhanced resolution of conserved and rearranged chromosomal segments presented in this study provides clarification of chromosome evolution history.