Mild digestion of Xenopus nuclei with micrococcal nuclease results in the cleavage of oocyte-type 5S RNA genes once every four nucleosomes, or about once per tandem repeating unit of 5S DNA. This specific cleavage pattern is observed with nuclei from somatic cells where oocyte-type 5S genes are never transcribed (blood and liver) and with cultured cell nuclei where these genes are in a DNAase I-sensitive chromatin conformation and low level transcription is observed. Cleavage of protein-free DNA with micrococcal nuclease does not result in a specific digestion pattern. The similarity of the nuclease-generated repeat length and the sequence repeat length of oocyte-type 5S genes suggested a sequence-specific arrangement of nucleosomes on these DNA sequences. Restriction endonuclease analysis indicates that micrococcal nuclease preferentially cleaves in a restricted region within the 5S repeating unit, about 200 bp from the single Hind III site. Using specific end-labeled DNA probes derived from cloned 5S DNA we can recognize at least four possible modes of organization of the nucleosomes on 5S DNA. In each of these phase arrangements, functionally significant regions of the 5S gene (start of transcription, middle control region and transcription termination site) are found in or near nucleosome linkers.