Chromatin repeat length is known to differ among the tissues of organisms. We have examined the repeat length of somatic cells in culture and have found an unexpected correlation between this parameter and the state of differentiation of the cells. Three sequentially derived subclones of a rat hepatoma, similar in phenotype, present a constant repeat length of 189 (+/- 1) base pairs. Three independent variant clones do not express hepatic functions and each has a different repeat length: 184 base pairs, 188 base pairs, and 192 base pairs. Somatic hybrids between the two types of cells in which liver functions are extinguished have a repeat length close or identical to that of the nonexpressing parent. In the case of one hybrid clone that reexpresses the liver phenotype and has undergone some chromosome loss, the 189-base-pair repeat length characteristic of the well-differentiated cells is reestablished. Finally, an amelanotic variant clone derived from a pigmented hamster melanoma clone also shows a signficantly altered repeat length. No correlations have been observed between repeat length and either generation time or chromosome number. Maintenance of the expression of differentiation therefore appears to be associated with a constant repeat length, and loss of the former with a modification in repeat length, suggesting that chromatin packaging is intimately involved in the regulation of gene expression.