Rat and human ribosomal RNA gene fragments in supercoiled plasmids were examined for S1 nuclease hypersensitivity. In the transcribed portion of genes the number and distribution of S1 sites were found to be species specific. No S1 sites were detected in the promoter regions. In the nontranscribed spacer (NTS), downstream of the 3' end of 28S RNA gene, S1 sites appear to be conserved in rat and human rDNAs.A rat NTS fragment (2987 nucleotides long), containing three S1 sites was sequenced and the SI sites in this region were localized in polypyrimidine.polypurine simple repeat sequences. Other types of simple sequences, two type2 Alu repeats and an ID sequence were also found in the sequenced region. The possible role of simple sequences and SI sites in transcription and in recombination events of rDNA is discussed.