We have constructed a genetic linkage map of the long arm of human chromosome 21 comprising 27 DNA markers. This map is an updated version of that reported earlier by group (1989, Genomics 4: 579-591), which contained 17 DNA markers. The current markers consist of 10 genes and 17 anonymous sequences. Traditional methods (restriction fragment length polymorphisms) were used to map 25 of these markers, whereas 2 markers were studied by polymerase chain reaction amplification of (GT)n dinucleotide repeats. Linkage analysis was performed on 40 CEPH families using the computer program packages LINKAGE, CRI-MAP, and MAPMAKER. Recombination rates were significantly different between the sexes, with the male map being 132 cM and the female map being 161 cM, assuming Kosambi interference and a variable ratio of sex difference in recombination. Approximately one-half of the crossovers in either sex occur distally, in terminal band 21q22.3, which also contains 16 of the markers studied. The average distance between adjacent markers was 6 cM.