In eukaryotic nuclei, DNA is wrapped around an octamer of core histones to form nucleosomes, and chromatin fibers are thought to be stabilized by linker histones of the H1 type. Higher eukaryotes express multiple variants of histone H1; chickens possess six H1 variants. Here, we generated and analyzed the phenotype of a complete deletion of histone H1 genes in chicken cells. The H1-null cells showed decreased global nucleosome spacing, expanded nuclear volumes, and increased chromosome aberration rates, although proper mitotic chromatin structure appeared to be maintained. Expression array analysis revealed that the transcription of multiple genes was affected and was mostly downregulated in histone H1-deficient cells. This report describes the first histone H1 complete knockout cells in vertebrates and suggests that linker histone H1, while not required for mitotic chromatin condensation, plays important roles in nucleosome spacing and interphase chromatin compaction and acts as a global transcription regulator.