The chromatins of rat liver and chicken reticulocytes contain a small RNA of identical length (29 nucleotides). This RNA species differs from the degradation products of nuclear RNA present in the chromatin in possessing a free 3'-hydroxyl terminus. It stays associated with chromatin and is not released from the nuclei during the extraction of nuclear ribonucleoprotein particles. This RNA species was purified from the third fraction of non-histone chromosomal proteins eluted from Sephadex G-200 (fraction 3 RNA). When fraction 3 RNA, isolated from rat liver, was used to screen a rat genomic library approximately 3% of the phage plaques hybridized with the RNA. DNA isolated from four randomly selected hybridizing phage clones hybridized with purified 29-nucleotide RNA demonstrating that the sequences present in the four clones represent those of the 29-nucleotide RNA species and not those of minor contaminants. Each of the four clones contained a different amount of sequences complementary to the RNA species 29-nucleotides-long and in each clone the DNA sequences homologous to this RNA were surrounded by different restriction sites. Hybridization of 3'-32P-labeled fraction 3 RNA with blots of total genomic DNA established that some sequences homologous to this RNA are dispersed in the DNA and others are present on one EcoRI restriction fragment, which is approximately 6000 base pairs long. Fraction 3 RNA is not a degradation product of ribosomal or transfer RNA, it is transcribed from middle repetitive DNA and probably originates by specific release during the formation and/or processing of high-molecular-weight transcripts. In normal liver cells its sequences are not released into the cytoplasm.