Histone H1 content and synthesis were examined in normal, Werner-syndrome, and transformed fibroblasts. Analysis of 3H-lysine incorporation indicated that senescent cells, but not G1-arrested young cells, had a lower ratio of molar synthesis of H1 histone to nucleosome histones than did growing young cells or gamma-ray-transformed cells. Furthermore, a biochemical study of histone H1 content plotted as a function of DNA synthesis activity and an immunocytological study using antiserum against histone H1 revealed that senescent cells had a lower histone H1 content than did young cultures at all stages of cell proliferation. Werner syndrome skin fibroblasts at early passage, however, had amounts of histone H1 comparable to those of age-matched normal control fibroblasts. We conclude that a decline, with increasing passage number, in content and synthesis of H1 histone relative to nucleosomal histones (Mitsui et al., 1980) was not simply due to passage-related accumulation of G1-arrested cells, but actually reflected age specific changes of cultured human fibroblasts. The depletion of histone H1 in the chromatin of senescent cells is a possible cause of DNA strand breakage or relaxation of gene repression.