Attention was focussed on the question whether the most active genes of the polytene genome of Chironomus tentans, i.e. rDNA and Balbiani rings, have undergone the same number of duplications as the bulk of the genome. RNA was extracted from isolated salivary glands, enriched for messenger RNA by poly U sepharose chromatography, and labelled with 125Iodine. In situ hybridization showed that, apart from rRNA, Balbiani ring 1 and 2 transcripts are the major hybridizing RNA species present in this enriched preparation. BR1 hybridized only at the b-region. Transcripts of BR6 and puff I 17B are shown to be salivary gland specific and hybridize in situ to an extent of less than 5% of the RNA hybridized to BR1 plus BR2. Saturation hybridization levels of rRNA and Balbiani ring (BR1 plus BR2) RNA with polytene DNA from salivary glands were determined and compared with the saturation levels measured for DNA from adult flies and larvae. rRNA hybridized to the same level (0.09%) with polytene DNA as with DNA from primarily diploid cells (adult fly DNA) or from larvae. An equal hybridization level (0.04%) with each of these three types of DNA was also obtained for BR RNA. The results demonstrate that, during polytenization in C. tentans, the genes for rRNA and BR1 and 2 are duplicated to the same extent as the bulk of the genome.