Down syndrome (DS; trisomy 21) is associated with a wide range of variable clinical features, one of the most common being congenital heart defects (CHD). We used molecular genetic techniques to study the inheritance of genes on chromosome 21 in children with DS and CHD. Polymorphic markers on the long arm of chromosome 21 were analysed in 99 families who had a child with DS. Of these, 60 children had a CHD and 39 children had no CHD. Heterotrisomy describes the inheritance of an allele from each of three different grandparents. In some cases heterotrisomy will involve the inheritance of three different alleles. Heterotrisomic regions were defined as those showing retention of non-disjoining parental heterozygosity at polymorphic loci in the non-disjoined chromosomes of children with DS. Using polymorphic non-coding markers, we identified a consistent 9.6-cM minimum region (D21S167-HMG14) of heterotrisomy in children with DS and ventricular septal defect (VSD). Comparing individuals with DS and VSD to all others with DS (those either with no CHD or with any other CHD combined) shows the individuals with DS and VSD to have significantly more non-reduction or heterotrisomy in this region (P=0.006, Fisher's exact test, two-tailed). We postulate that heterotrisomy for a gene or genes in this region is a contributing factor to the pathogenesis of VSD in trisomy 21 either through the presence of three different specific alleles or through the presence of specific combinations of alleles.