The organization of the human Surfeit locus containing the six sequence-unrelated housekeeping genes Surf-1 to Surf-6 (HGMW-approved symbols SURF1-SURF6) has been determined. The human surfeit locus occupies about 60 kb of DNA, and the tightly clustered gene organization and the juxtaposition of the human genes are similar to the mouse and chicken surfeit loci with the 5' end of each gene associated with a CpG-rich island. Whereas in the mouse the Surf-2 and Surf-4 genes overlap at their 3' ends, the human Surf-2 and Surf-4 genes have been found to be separated by 302 bp due to a much shorter 3' untranslated region in the human Surf-2 gene. The distance between the 3' ends of the human Surf-1 and Surf-3 genes is 374 bp, and the distance between the 5' ends of the human Surf-3 and Surf-5 genes is only 112 bp. Unusually the human Surf-5 gene contains an intron in its 5' untranslated region not found in the mouse or rat Surf-5 genes. This additional intron is also found in the Surf-5 gene of both Old and New World monkeys, being generated before the divergence of human and prosimians but after the divergence of primates and rodents. A contig of 200 kb containing the human Surfeit locus has been constructed from overlapping cosmid, P1, and PAC clones. Approximately 40 kb proximal to the 3' end of the Surf-6 gene, the 5' region of the ABO glycosyltransferase gene has been detected. This allows us to determine the orientation of the Surfeit and ABO loci with respect to each other and to the telomere and centromere of human chromosome 9.