pCtBR2-1 is a recombinant plasmid with a 750-bp insert of Chironomus tentans genomic DNA. When pCtBR2-1 was hybridized in situ to salivary gland polytene chromosomes, it hybridized exclusively to Balbiani ring 2 (BR2), a giant chromosomal puff. It was also shown that the insert contained four tandemly repeated sequences that were delineated by HinfI sites which occurred every 190 bp. The purified insert reassociated to C. tentans DNA with a C0t1/2 = 0.48 indicating that the sequence was moderately repeated within the genome. Hybridization of radioactive pCtBR2-1 to nitrocellulose blots containing partial HinfI digests of genomic DNA revealed that the 190-bp repeats were organized into one or more blocks of 11 to 12 copies in tandem. Hybridization of the recombinant plasmid to limit digests of genomic DNA also demonstrated that repeated sequences in BR2 were not homogeneous. As much as 70% of BR2 appeared to be represented by a 26-kb HhaI-resistant core, while the remaining 30% may have HhaI sites at 190-bp intervals, similar to pCtBR2-1.