Segregation analysis of CEPH and other pedigrees yielded six paternal crossover breakpoints in the approximately 85 kb interval between the minisatellite loci D16S309 (MS205) and D16S83 (EKMDA2) in 16p13.3. Three crossovers were mapped to within the same small (<3 kb) interval, which does not co-localize with any tandem repeat array or expressed sequence. Haplotyping of loci harbouring single nucleotide polymorphism (SNP) markers in this interval confirmed the exchange of flanking markers in the three recombinant individuals. Sequence analysis revealed the presence of recombination-associated motifs and binding sites for the protein translin. Haplotyping of 108 individuals from three European populations at four loci harbouring SNPs showed substantial linkage equilibrium across this interval. Hence molecular and population genetic data are consistent with the presence of an intense male-specific recombination hotspot at this locus.