The mouse His-1 gene encodes a spliced and polyadenylated RNA with no long open reading frame (ORF), making it difficult to distinguish a functional protein coding domain. To identify candidate protein coding ORFs, and other functionally significant regions, we have isolated and sequenced 8.5 kb of a human genomic DNA that is homologous to the mouse His-1 gene. Alignment of the mouse and human sequences required no extensive gapping, indicating that evolutionary constraints have maintained a requirement for colinearity in genomic organization. We have identified the mouse transcriptional start point (tsp) and shown that the sequence of the 5'-flanking region is highly conserved in the human homolog. Sequence comparisons between the mouse and human genes identified conservation of other putative functional domains in exon 3 and in each of the two introns. Southern blot analysis with probes from each of these regions detected homologs in multiple other vertebrate species. However, none of the multiple candidate ORFs in the mouse RNA were conserved in the human sequence, suggesting that the RNA is unlikely to encode a protein. These data suggest that the RNA may be the final and functional product from the mouse His-1 gene.