The human beta-globin gene complex spans a region of 70 kb and contains numerous sequence variants. These variant sites form a 5' cluster (5' beta-haplotype) and a 3' cluster (3' beta-haplotype) with strong linkage disequilibrium among the sites within each cluster, but not between the two clusters. The 9-kb region between the 5' and 3' clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a 'hotspot' of recombination. We describe three families with evidence of meiotic recombination within this 'hotspot' of the beta-globin gene cluster and in which the cross-over breakpoints have been defined at the sequence level. In one family, the recombination has occurred in the maternal chromosome within a region of 361 bp between positions -911 and -550 5' to the beta-globin gene. In the other two families, the recombination has occurred in the paternal chromosome within a region of approximately 1,100 bp between positions -542 and +568 relative to the beta-globin gene cap site. Both regions occur within the 2-kb region of replication initiation (IR) in the beta-globin gene domain with no overlap. The IR region contains a consensus sequence for a protein (Pur), which binds preferentially to single-stranded DNA, a role implicated in recombination events.