Chromatin was prepared from isolated nuclei of proliferating and differentiated cultures of C1300 mouse neuroblastoma cells. Differentiation was induced by serum withdrawal or treatment with dibutyryl cyclic AMP. The ability to support DNA-dependent RNA synthesis when assayed in a cell-free system is three times greater for chromatin from proliferating cells. Histones isolated from proliferating and differentiated cells were fractionated electrophoretically. The relative amounts of proteins present in the five major histone fractions were similar. In contrast, there were significant differences in the nonhistone chromosomal proteins synthesized and associated with the genome of proliferating and differentiating neuroblastoma cells. Such differences are reflected by modifications in the electrophoretic banding patterns and in incorporation of [3H]tryptophan into various molecular weight classes of nonhistone chromosomal polypeptides. A functional relationship between changes in the nonhistone chromosomal proteins and variations in the transcriptional activity accompanying differentiation of neuroblastoma cells may exist.