The 5S RNA gene cluster of Drosophia melanogaster is a tandem array of a repeat unit made up of a 135 bp gene plus a 238 bp spacer. The length of the 5S repeat (373 bp) equals the average length of a Drosophila dinucleosome. Digestion of Drosophila nuclei with micrococcal nuclease generates discrete 5S RNA gene subfragments when the purified DNA is further cleaved with a single-cut restruction enzyme. We have mapped four micrococcal nuclease-sensitive sites within the 5S repeat: A1, centered at bp--110 (+1 being the G:C bp at the start of the gene); A1', at bp--80; A2, at bp +80, within the intragenic control region; and B, at --190 bp. These findings suggest that nucleosomes can be positioned on the 5S gene repeat in one of two possible phases, A or B. In the A phase a potential regulatory sequence near the center of the gene is exposed in one of the two linkers of the repeat. In the B phase, in contrast, one of the linkers includes the 5' end of the gene.