A technique for isolating DNA sequences that are likely to be transcriptionally active in both humans and mice has been developed. This method is based on the screening of a human genomic DNA library with single stranded cDNA prepared from late gestation mouse fetal RNA. Using a human chromosome 21 DNA library, we have isolated and characterized two clones which are entirely composed of single copy human DNA sequences and are 2.7 and 6.7 kilobases in length. The 2.7 kilobase clone is homologous to a transcript found predominantly in nonpolyadenylated human fibroblast RNA. It also shows homology to mouse genomic DNA and recognises several polyadenylated RNAs in mouse testis. This cloned sequence has been mapped by in situ hybridization to the distal third of chromosome 21, the region involved in the causation of Down syndrome.