The human HMG-14 multigene family is one of the largest retropseudogene families known. To identify and isolate a functional human HMG-14 gene, genomic clones, selected with the cDNA, were screened with a set of 6 oligonucleotides. A single genomic clone was isolated suggesting that the human genome contains few, and perhaps only one, functional genes. An 8882-base pair (bp) genomic clone containing the complete, 6804-bp-long human gene together with 850 bp 5' to the start of transcription and 1228 bp 3' to the end of transcription was sequenced. The gene is comprised of 6 exons ranging in size from 30 to 839 bp, two of which code for the entire DNA binding site of the protein, and has several features typical of "housekeeping" genes. Using human-rodent somatic cell hybrids, the HMG-14 gene was localized to human chromosome 21. A restriction fragment length polymorphism, useful for further analysis and mapping, has been detected. The present article, which describes the first isolation and characterization of a gene coding for chromosomal protein HMG-14, indicates that genes coding for HMG-14 and HMG-17 may share several distinctive characteristics. Comparison with the human and chicken HMG-17 genes reveals that all contain 6 exons, that all have exons of similar size, that all have 5' regions highly enriched in GC residues and that all have features typical of housekeeping genes.