We describe a cloned segment of D. melanogaster DNA (cDm219) that contains five tandemly arranged sequence units homologous to the type I insertion sequence found in the majority of 28S rRNA genes on the X chromosome. Heteroduplex studies show that two of the units have a deletion corresponding to a 1.1 kb piece of DNA close to the right-hand end of the type I insertion. Another unit has a 7.5 kb sequence (zeta) substituted for a 0.95 kb piece of DNA close to the left-hand part of the type I rDNA insertion. The two remaining units are interrupted by the Col E1 plasmid vector. There are also differences in the restriction endonuclease cleavage maps both between the units of cDm219 themselves and compared to the restriction endonuclease cleavage maps of cloned rDNA segments that contain type I insertions. Quantitation of the gel transfer hybridization of zeta element probes to restriction endonuclease digests of D. melanogaster DNA indicates there are 30–40 copies of zeta sequences distributed in seven major arrangements within the haploid genome. The hybridization of zeta and insertion sequence probes to a library of D. melanogaster DNA segments cloned in bacteriophage λ indicates at least 4–6 copies of the zeta element could be linked to insertion sequences. The common site of in situ hybridization of ζ sequences is to the chromocentral heterochromatin of polytene chromosomes.