The H1 histone family in mammals contains at least seven subtypes. In the past we have isolated six of the seven genes encoding these isoforms. To complete the set of the human H1 histone genes, we have designed two PCR primers deduced from a partially published sequence of the remaining histone H1 gene [Carozzi et al. (1984) Science 224, 1115-1118] and from a consensus sequence which we have derived from the conserved region of human histone H1 genes. Using these primers we have amplified a 417-bp DNA fragment from total human DNA. This fragment was used for screening a human phage genomic library. Two overlapping clones were isolated. The region contains a set of 5 genes representing each of the five histone classes. In continuation of our numbering of human H1 genes, we have named this H1 gene H1.5. This gene encodes a protein almost identical to the previously published protein sequence designated H1a [Ohe et al. (1986) J. Biochem. 100, 359-368]; since the changes are in a region of some uncertainty of the peptide sequencing, we conclude that the newly isolated gene codes for the H1a protein. The structures of the flanking regions of the genes except the H2B gene are typical for histone genes. They include: (1) a CCAAT element in the promotor region, (2) a TATA box and (3) a palindromic termination element. The H2B sequence shows no typical regulatory elements and no complete ORF, therefore we consider it as a pseudogene. The expression of the H1.5 gene was examined in several cell lines.