Thirty-nine of the 44 chicken histone genes are located in a major gene cluster of 110 kb, the others being distributed in four separate regions. All 6 H1 genes, which are present in the cluster and encode different variants, are expressed in the DT40 chicken B cell line, at levels ranging from about 5 to 40%. To clarify differences in the natures of these H1 variants, using gene-targeting techniques, we generated a series of DT40 mutants, which are devoid of each of the 5 H1 genes, respectively. Analyses of six H1-deficient mutants, comprising the latter five and a previously generated H1-deficient mutant, revealed that the protein patterns on ED-PAGE were definitely different from each other, indicating that each ill variant plays an individual role in the transcription regulation of specific genes in DT40 cells.