Isolated, deproteinized nucleic acids from calf thymus and rat liver nuclei were fractionated on Bio-Gel A-5m into DNA and RNA fractions. Extraction of deproteinized DNA and nuclear RNA with 80% ethanol at pH 9.5 yielded low molecular weight peptides (deprimerones) capable of inhibiting DNA transcription in a cell-free system. The extracted peptidic fractions were further fractionated on a Sephadex G-25 column and collected as a fraction of mol. wt. between 1 500 and 600. The yield of DNA deprimerones was about 30 microgram/mg DNA and of nuclear RNA deprimerones 150-200 microgram/mg RNA (measured by amino acid composition). The nucleoplasm contained a negligible amount of free, soluble peptides (about 18 microgram/g tissue). The DNA and RNA deprimerones were characterized by their amino acid composition and by two-dimensional thin layer chromatography on cellulose gel plates which yielded two major and two minor fractions. DNA deprimerones isolated in this way are very similar, if not identical, to the affinity DNA deprimerones isolated from a nuclear extract on DNA-cellulose but differ from RNA deprimerones. DNA and RNA deprimerones were also compared with the nuclear deprimerones extracted directly from the lysed nuclei. Their amino acid composition is different from both DNA and nuclear RNA deprimerones. Two-dimensional thin layer chromatography on cellulose plates yielded about ten fractions (spots) with various amino acid composition and inhibitory activity for cell-free transcription.