Exon trapping was performed with chromosome 21 cosmids to identify those that may be involved in the pathogenesis of Down syndrome, or several of the genetic diseases that map to chromosome 21. BLASTX analysis revealed two exons with significant homology to a zebrafish protein (ES1) and an Escherichia coli protein (sigma cross-reacting protein 27A), both of unknown function. The exons also showed identity with several expressed sequence tags (ESTs). Sequences from all ESTs derived from this gene and reverse transcription-polymerase chain reaction (RT-PCR) analysis were used to determine the full cDNA sequence, which corresponded to an mRNA of 1.7 kb with an open reading frame of 268 amino acids. The mRNA from this gene, termed HES1, is ubiquitously expressed, but strongly so in heart and skeletal muscle. Potential mitochondrial targeting signals were found in both the human and zebrafish proteins, consistent with the high expression levels in muscle tissues. The strong homology between the E. coli, zebrafish and HES1 proteins suggests an important biological role. Hybridization of RT-PCR products to a cosmid contig in chromosome 21q22.3, mapped HES1 just proximal to D21S25, a critical mapping region for several genetic diseases. Given the mapping position, this gene is a candidate for involvement in these disorders, including autoimmune polyglandular disease type I and the autosomal nonsyndromic deafness loci, DFNB8 and DFNB10. In addition, the initial method of EST identification for gene isolation presented here is valid for many genes and can be used to obtain initial sequence contigs without cloning or library screening.