Using pulsed-field gel electrophoresis (PFGE), we have generated a 3-megabase (Mb) physical map of Xq28, a region of the human genome known to contain many disease loci. We have determined the location of the genes for protan/deutan colorblindness (R/GCP), factor VIII (F8), glucose-6-phosphate dehydrogenase (G6PD), and a series of RFLPs and have derived the following order for this region: R/GCP-GdX-G6PD-F8-DXS115-DXS33-DXS134-D XS15. Using newly isolated probes, we have also established the direction of transcription of F8 within the map and have localized CpG islands flanking this gene.