1Department of Biomedicine and Surgery, Division of Cell Biology, Faculty of Health Sciences, Linköpings universitet, Sweden and 2Department of Medical Chemistry and Biochemistry, University of Innsbruck, Austria Histone H1 binds to the nucleosome at the entry and exit point of the DNA from the nucleosome core, as well as to linker DNA. The human histone H1 family consists of seven subtypes, H1.1-H1.5, H1 and H1t, all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails of high positive charge. In this study, a hydrophilic-interaction liquid chromatographic method was used for detecting sequence variations within the subtypes. Two sequence variants of wild type H1 sequences were found. In K562 erythroleukemic cells, alanine in position 17 of H1.2 was substituted by valine, and in Raji B-lymphoblastoid cells lysine in position 173 of H1.4 was replaced by arginine. DNA sequencing of the corresponding gene segments confirmed these findings. In K562 cells, a homozygous GCC>GTC shift at codon 18 was detected, causing the H1.2 Ala17Val sequence variant, since the initial methionine is removed in H1 histones. In Raji cells, a heterozygous AAA>AGA codon change at position 174 in H1.4 was found, giving rise to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal population was determined. An RFLP-assay was developed for detection of the H1.2 GCC>GTC shift at codon 18, and the allele frequency was determined to be 6.8%, indicating that this is a relatively frequent polymorphism. The AAA>AGA codon change at position 174 in H1.4 was detected using denaturing HPLC. This sequence variation was only found in Raji cells and it was not present in a normal population, or in six other cell lines derived from individuals suffering from Burkitt’s lymphoma. The significance of these sequence variants is unclear, but binding characteristics of the deviating histones H1.2 and H1.4 may be altered, and thereby processes like chromatin higher order organization and epigenetic control of gene regulation can be affected.