The distribution of RNA polymerase B (or II) in native and fixed polytene chromosomes isolated from salivary glands of Chironomus tentans and C. pallidivittatus was investigated by both indirect immunofluorescence and autoradiography. The chromosomes, especially the Balbiani rings (BR2, BR1 and BR3), were examined during periods of stimulated and repressed RNA synthesis. In repressed BR2a and, after the salivary gland chromosomes had been stretched, in various chromosomal segments, it was possible to establish unequivocally that RNA polymerase B is not confined to puffs, but also occurs in interbands. The enzyme was absent from the bands, or at least there was not enough of it to be detected with indirect immunofluorescence. It was shown that the distribution of the indirect immunofluorescence in the chromosomes concurs with that of the 3H-uridine or 3H-UTP labeling. However, RNA polymerase B molecules remain associated with the chromosomal template even after an in vivo alpha-amanitin or actinomycin D treatment to inhibit RNA synthesis. Following heat shocks (37 degrees C to 39 degrees C), transcriptively active RNA polymerase B is still found in interbands, in the BRs and in other puffs that have collapsed as a result of the heat treatment; the greatest enzyme concentrations, however, are in the stimulated heat-shock puffs.