To explore the extent to which the X chromosome has been conserved during mammalian evolution, we compared six loci that are X-linked in the human genome with the corresponding genes of the North American marsupial, the Virginia opossum (Didelphis virginiana). Our analysis shows that in the opossum genome there are sequences highly homologous to those of human cDNAs for housekeeping genes, glucose-6-phosphoribosyltransferase (HPRT), phosphoglycerate kinase A (PGK1), and alpha-galactosidase A (GLA). However, ornithine transcarbamylase and blood clotting Factor IX--tissue-specific genes that are X-linked in eutherians mammals--have no highly conserved homologs in the marsupial genome. By cloning opossum G6PD and HPRT, we found that these genes are X-linked in the opossum and that homologous sequences are limited to coding regions. As all genomic fragments hybridizing with the human GLA probe show dosage effects, it is likely that the opossum counterpart is X-linked. Finally, the pattern of hybridization suggests that the autosomal pseudogenes of HPRT and PGK1 in the opossum have remained highly homologous to the human X-linked genes.